Expanding the eukaryotic genetic code

ABSTRACT

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/825,867, filed Apr. 16, 2004 now U.S. Pat. No. 7,618,775, whichclaims the benefit of application U.S. Ser. No. 60/463,869 entitled“Expanding the Eukaryotic Genetic Code” by Chin et al., filed Apr. 17,2003; U.S. Ser. No. 60/479,931 entitled “Expanding the EukaryoticGenetic Code” by Chin et al. filed Jun. 18, 2003; U.S. Ser. No.60/493,014 entitled “Expanding the Eukaryotic Genetic Code” by Chin etal., filed Aug. 5, 2003; and U.S. Ser. No. 60/496,548 entitled“Expanding the Eukaryotic Genetic Code” by Chin et al., filed Aug. 19,2003. Priority to and benefit of each of these prior applications ishereby claimed.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No. GM 62159from the National Institutes of Health and support under GrantDE-FG0300ER45812 from the Department of Energy. The government hascertain rights to this invention.

FIELD OF THE INVENTION

The invention pertains to the field of translation biochemistry ineukaryotic cells. The invention relates to methods for producing andcompositions of orthogonal tRNAs, orthogonal synthetases and pairsthereof, in eukaryotic cells. The invention also relates to compositionsof unnatural amino acids, proteins and methods of producing proteins ineukaryotic cells that include unnatural amino acids.

BACKGROUND OF THE INVENTION

The genetic code of every known organism, from bacteria to humans,encodes the same twenty common amino acids. Different combinations ofthe same twenty natural amino acids form proteins that carry outvirtually all the complex processes of life, from photosynthesis tosignal transduction and the immune response. In order to study andmodify protein structure and function, scientists have attempted tomanipulate both the genetic code and the amino acid sequence ofproteins. However, it has been difficult to remove the constraintsimposed by the genetic code that limit proteins to twenty geneticallyencoded standard building blocks (with the rare exception ofselenocysteine (see, e.g., A. Bock et al., (1991), MolecularMicrobiology 5:515-20) and pyrrolysine (see, e.g., G. Srinivasan, etal., (2002), Science 296:1459-62).

Some progress has been made to remove these constraints, although thisprogress has been limited and the ability to rationally control proteinstructure and function is still in its infancy. For example, chemistshave developed methods and strategies to synthesize and manipulate thestructures of small molecules (see, e.g., E. J. Corey, & X.-M. Cheng,The Logic of Chemical Synthesis (Wiley-Interscience, New York, 1995)).Total synthesis (see, e.g., B. Merrifield, (1986), Science 232:341-7(1986)), and semi-synthetic methodologies (see, e.g., D. Y. Jackson etal., (1994) Science 266:243-7; and, P. E. Dawson, & S. B. Kent, (2000),Annual Review of Biochemistry 69:923-60), have made it possible tosynthesize peptides and small proteins, but these methodologies havelimited utility with proteins over 10 kilo Daltons (kDa). Mutagenesismethods, though powerful, are restricted to a limited number ofstructural changes. In a number of cases, it has been possible tocompetitively incorporate close structural analogues of common aminoacids throughout proteins. See, e.g., R. Furter, (1998), Protein Science7:419-26; K. Kirshenbaum, et al., (2002), Chem Bio Chem 3:235-7; and, V.Doring et al., (2001), Science 292:501-4.

In an attempt to expand the ability to manipulate protein structure andfunction, in vitro methods using chemically acylated orthogonal tRNAswere developed that allowed unnatural amino acids to be selectivelyincorporated in response to a nonsense codon, in vitro (see, e.g., J. A.Ellman, et al., (1992), Science 255:197-200). Amino acids with novelstructures and physical properties were selectively incorporated intoproteins to study protein folding and stability and biomolecularrecognition and catalysis. See, e.g., D. Mendel, et al., (1995), AnnualReview of Biophysics and Biomolecular Structure 24:435-462; and, V. W.Cornish, et al. (Mar. 31, 1995), Angewandte Chemie-International Editionin English 34:621-633. However, the stoichiometric nature of thisprocess severely limited the amount of protein that could be generated.

Unnatural amino acids have been microinjected into cells. For example,unnatural amino acids were introduced into the nicotinic acetylcholinereceptor in Xenopus oocytes (e.g., M. W. Nowak, et al. (1998), In vivoincorporation of unnatural amino acids into ion channels in Xenopusoocyte expression system, Method Enzymol. 293:504-529) by microinjectionof a chemically misacylated Tetrahymena thermophila tRNA (e.g., M. E.Saks, et al. (1996), An engineered Tetrahymena tRNAGln for in vivoincorporation of unnatural amino acids into proteins by nonsensesuppression, J. Biol. Chem. 271:23169-23175), and the relevant mRNA.This has allowed detailed biophysical studies of the receptor in oocytesby the introduction of amino acids containing side chains with uniquephysical or chemical properties. See, e.g., D. A. Dougherty (2000),Unnatural amino acids as probes of protein structure and function, Curr.Opin. Chem. Biol. 4:645-652. Unfortunately, this methodology is limitedto proteins in cells that can be microinjected, and because the relevanttRNA is chemically acylated in vitro, and cannot be re-acylated, theyields of protein are very low.

To overcome these limitations, new components were added to the proteinbiosynthetic machinery of the prokaryote Escherichia coli (E. coli)(e.g., L. Wang, et al., (2001), Science 292:498-500), which allowedgenetic encoding of unnatural amino acids in vivo. A number of new aminoacids with novel chemical, physical or biological properties, includingphotoaffinity labels and photoisomerizable amino acids, keto aminoacids, and glycosylated amino acids have been incorporated efficientlyand with high fidelity into proteins in E. coli in response to the ambercodon, TAG, using this methodology. See, e.g., J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), Chem Bio Chem 11:1135-1137; J. W. Chin,et al., (2002), PNAS United States of America 99:11020-11024: and, L.Wang, & P. G. Schultz, (2002), Chem. Comm., 1-10. However, thetranslational machinery of prokaryotes and eukaryotes are not highlyconserved; thus, components of the biosynthetic machinery added to E.coli cannot often be used to site-specifically incorporate unnaturalamino acids into proteins in eukaryotic cells. For example, theMethanococcus jannaschii tyrosyl-tRNA synthetase/tRNA pair that was usedin E. coli is not orthogonal in eukaryotic cells. In addition, thetranscription of tRNA in eukaryotes, but not in prokaryotes, is carriedout by RNA Polymerase III and this places restrictions on the primarysequence of the tRNA structural genes that can be transcribed ineukaryotic cells. Moreover, in contrast to prokaryotic cells, tRNAs ineukaryotic cells need to be exported from the nucleus, where they aretranscribed, to the cytoplasm, to function in translation. Finally, theeukaryotic 80S ribosome is distinct from the 70S prokaryotic ribosome.Thus, there is a need to develop improved components of the biosyntheticmachinery to expand the eukaryotic genetic code. This invention fulfillsthese and other needs, as will be apparent upon review of the followingdisclosure.

SUMMARY OF THE INVENTION

The invention provides eukaryotic cells with translation components,e.g., pairs of orthogonal aminoacyl-tRNA synthetases (O-RSs) andorthogonal tRNAs (O-tRNAs) and individual components thereof, that areused in eukaryotic protein biosynthetic machinery to incorporate anunnatural amino acid in a growing polypeptide chain, in a eukaryoticcell.

Compositions of the invention include a eukaryotic cell (e.g., a yeastcell (such as a Saccharomyces cerevisiae cell), a mammalian cell, aplant cell, an algae cell, a fungal cell, an insect cell, etc.)comprising an orthogonal aminoacyl-tRNA synthetase (O-RS) (e.g., derivedfrom a non-eukaryotic organism, such as Escherichia coli, Bacillusstearothermophilus, etc.), where the O-RS preferentially aminoacylatesan orthogonal tRNA (O-tRNA) with at least one unnatural amino acid inthe eukaryotic cell. Optionally, two or more OtRNAs can be aminoacylatedin a given eukaryotic cell. In one aspect, an O-RS aminoacylates anO-tRNA with the unnatural amino acid, e.g., at least 40%, at least 45%,at least 50%, at least 60%, at least 75%, at least 80%, or even 90% ormore as efficiently as does an O-RS having an amino acid sequence, e.g.,as set forth in SEQ ID NO.: 86 or 45. In one embodiment, an O-RS of theinvention aminoacylates the O-tRNA with the unnatural amino acid, e.g.,at least 10-fold, at least 20-fold, at least 30-fold, etc., moreefficiently than the O-RS aminoacylates the O-tRNA with a natural aminoacid.

In one embodiment, the O-RS or a portion thereof is encoded by apolynucleotide sequence as set forth in any one of SEQ ID NO.: 3-35(e.g., 3-19, 20-35, or any other subset of sequences 3-35), or acomplementary polynucleotide sequence thereof. In another embodiment,the O-RS comprises an amino acid sequence as set forth in any one of SEQID NO.: 36-63 (e.g., 36-47, 48-63, or any other subset of 36-63), and/or86, or a conservative variation thereof. In yet another embodiment, theO-RS comprises an amino acid sequence that is, e.g., at least 90%, atleast 95%, at least 98%, at least 99%, or at least 99.5% or more,identical to that of a naturally occurring tyrosyl aminoacyl-tRNAsynthetase (TyrRS) and comprises two or more amino acids from groupsA-E. Group A includes valine, isoleucine, leucine, glycine, serine,alanine, or threonine at a position corresponding to Tyr37 of an E. coliTyrRS. Group B includes aspartate at a position corresponding to Asn126of an E. coli TyrRS. Group C includes threonine, serine, arginine,asparagine or glycine at a position corresponding to Asp182 of an E.coli TyrRS. Group D includes methionine, alanine, valine, or tyrosine ata position corresponding to Phe183 of an E. coli TyrRS; and, group Eincludes serine, methionine, valine, cysteine, threonine, or alanine ata position corresponding to Leu186 of an E. coli TyrRS.

Any subset of combinations of these groups are a feature of theinvention. For example, in one embodiment, the O-RS has two or moreamino acids selected from valine, isoleucine, leucine, or threonineoccurs at a position corresponding to Tyr37 of E. coli TyrRS; threonine,serine, arginine, or glycine at a position corresponding to Asp182 of E.coli TyrRS; methionine, or tyrosine at a position corresponding toPhe183 of E. coli TyrRS; and, serine, or alanine at a positioncorresponding to Leu186 of E. coli TyrRS. In another embodiment, theO-RS includes two more amino acids selected from glycine, serine, oralanine at a position corresponding to Tyr37 of E. coli TyrRS, aspartateat a position corresponding to Asn126 of E. coli TyrRS, asparagine at aposition corresponding to Asp182 of E. coli TyrRS, alanine, or valine,at a position corresponding to Phe183 of E. coli TyrRS, and/ormethionine, valine, cysteine, or threonine, at a position correspondingto Leu186 of E. coli TyrRS.

In another embodiment, the O-RS has one or more improved or enhancedenzymatic properties for the unnatural amino acid as compared to anatural amino acid. For example, the improved or enhanced properties forthe unnatural amino acid as compared to a natural amino acid include anyof, e.g., a higher Km, a lower Km, a higher kcat, a lower kcat, a lowerkcat/km, a higher kcat/km, etc.

The eukaryotic cell also optionally includes an unnatural amino acid(s).The eukaryotic cell optionally includes an orthogonal tRNA (O-tRNA)(e.g., derived from a non-eukaryotic organism, such as Escherichia coli,Bacillus stearothermophilus, and/or the like), where the O-tRNArecognizes a selector codon and is preferentially aminoacylated with theunnatural amino acid by the O-RS. In one aspect, the O-tRNA mediates theincorporation of the unnatural amino acid into a protein with, e.g., atleast 45%, at least 50%, at least 60%, at least 75%, at least 80%, atleast 90%, at least 95%, or 99% or the efficiency of a tRNA thatcomprises or is processed in a cell from a polynucleotide sequence asset forth in SEQ ID NO.: 65. In another aspect, the O-tRNA comprises thesequence of SEQ ID NO.:65, and the O-RS comprises a polypeptide sequenceselected from an amino acid sequence set forth in any one of SEQ ID NO.:36-63 (e.g., 36-47, 48-63, or any other subset of 36-63), and/or 86,and/or a conservative variation thereof.

In another embodiment, the eukaryotic cell comprises a nucleic acid thatcomprises a polynucleotide that encodes a polypeptide of interest, wherethe polynucleotide comprises a selector codon that is recognized by theO-tRNA. In one aspect, the yield of the polypeptide of interestcomprising the unnatural amino acid is, e.g., at least 2.5%, at least5%, at least 10%, at least 25%, at least 30%, at least 40%, 50% or more,of that obtained for the naturally occurring polypeptide of interestfrom a cell in which the polynucleotide lacks the selector codon. Inanother aspect, the cell produces the polypeptide of interest in theabsence of the unnatural amino acid, with a yield that is, e.g., lessthan 35%, less than 30%, less than 20%, less than 15%, less than 10%,less than 5%, less than 2.5%, etc., of the yield of the polypeptide inthe presence of the unnatural amino acid.

The invention also provides a eukaryotic cell comprising an orthogonalaminoacyl-tRNA synthetase (O-RS), an orthogonal tRNA (O-tRNA), anunnatural amino acid, and a nucleic acid that comprises a polynucleotidethat encodes a polypeptide of interest. The polynucleotide comprises aselector codon that is recognized by the O-tRNA. In addition, the O-RSpreferentially aminoacylates the orthogonal tRNA (O-tRNA) with theunnatural amino acid in the eukaryotic cell, and the cell produces thepolypeptide of interest in the absence of the unnatural amino acid, witha yield that is, e.g., less than 30%, less than 20%, less than 15%, lessthan 10%, less than 5%, less than 2.5%, etc., of the yield of thepolypeptide in the presence of the unnatural amino acid.

Compositions that include a eukaryotic cell comprising an orthogonaltRNA (O-tRNA) are also a feature of the invention. Typically, the O-tRNAmediates incorporation of an unnatural amino acid into a protein that isencoded by a polynucleotide that comprises a selection codon that isrecognized by the O-tRNA in vivo. In one embodiment, the O-tRNA mediatesthe incorporation of the unnatural amino acid into the protein with,e.g., at least 45%, at least 50%, at least 60%, at least 75%, at least80%, at least 90%, at least 95%, or even 99% or more the efficiency of atRNA that comprises or is processed in a cell from a polynucleotidesequence as set forth in SEQ ID NO.: 65. In another embodiment, theO-tRNA comprises or is processed from a polynucleotide sequence as setforth in SEQ ID NO.: 65, or a conservative variation thereof. In yetanother embodiment, the O-tRNA comprises a recyclable O-tRNA.

In one aspect of the invention, the O-tRNA is post-transcriptionallymodified. The invention also provides a nucleic acid that encodes anO-tRNA in a eukaryotic cell, or a complementary polynucleotide thereof.In one embodiment, the nucleic acid comprises an A box and a B box.

The invention also features methods of producing translationalcomponents, e.g., O-RSs or O-tRNA/O-RS pairs (and translationalcomponents produced by these methods). For example, the inventionprovides methods of producing an orthogonal aminoacyl-tRNA synthetase(O-RS) that preferentially aminoacylates an orthogonal tRNA with anunnatural amino acid in a eukaryotic cell. The method includes, e.g.,(a) subjecting to positive selection, in the presence of an unnaturalamino acid, a population of eukaryotic cells of a first species, wherethe eukaryotic cells each comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker; where cellsthat survive the positive selection comprise an active RS thataminoacylates the orthogonal tRNA (O-tRNA) in the presence of anunnatural amino acid. The cells that survive the positive selection aresubjected to negative selection in the absence of the unnatural aminoacid to eliminate active RSs that aminoacylate the O-tRNA with a naturalamino acid. This provides the O-RS that preferentially aminoacylates theO-tRNA with the unnatural amino acid.

In certain embodiments, the polynucleotide that encodes the positiveselection marker is operably linked to a response element and the cellsfurther comprise a polynucleotide that: a) encodes a transcriptionalmodulator protein (e.g., a eukaryotic transcriptional modulator protein,etc.) that modulates transcription from the response element, and b)comprises at least one selector codon. The incorporation of theunnatural amino acid into the transcriptional modulator protein by theO-tRNA aminoacylated with the unnatural amino acid results intranscription of the positive selection marker. In one embodiment, thetranscriptional modulator protein is a transcriptional activator protein(e.g., GAL4, etc.), and the selector codon is an amber stop codon, e.g.,where the amber stop codon is located in or substantially near a portionof the polynucleotide that encodes a DNA binding domain of thetranscriptional activator protein.

The positive selection marker can be any of a variety of molecules. Inone embodiment, the positive selection marker comprises a nutritionalsupplement for growth and the selection is performed on a medium thatlacks the nutritional supplement. In another embodiment, thepolynucleotide that encodes the positive selection marker is, e.g., anura3, leu2, lys2, lacZ gene, his3 (e.g., where the his3 gene encodes animidazole glycerol phosphate dehydratase, detected by providing3-aminotriazole (3-AT)), and/or the like. In yet another embodiment, thepolynucleotide that encodes the positive selection marker comprises aselector codon.

As with the positive selection marker, the negative selection marker canalso be any of a variety of molecules. In certain embodiments, thepolynucleotide that encodes the negative selection marker is operablylinked to a response element from which transcription is mediated by thetranscriptional modulator protein. The incorporation of a natural aminoacid into the transcriptional modulator protein by the O-tRNAaminoacylated with a natural amino acid results in transcription of thenegative selection marker. In one embodiment, the polynucleotide thatencodes the negative selection marker is, e.g., an ura3 gene and thenegative selection is accomplished on a medium that comprises5-fluroorotic acid (5-FOA). In another embodiment, the medium used fornegative selection comprises a selecting or screening agent that isconverted to a detectable substance by the negative selection marker. Inone aspect of the invention, the detectable substance is a toxicsubstance. In one embodiment, the polynucleotide that encodes thenegative selection marker comprises a selector codon.

In certain embodiments, the positive selection marker and/or thenegative selection marker comprises a polypeptide that fluoresces orcatalyzes a luminescent reaction in the presence of a suitable reactant.In one aspect of the invention, the positive selection marker and/or thenegative selection marker is detected by fluorescence-activated cellsorting (FACS), or by luminescence. In certain embodiments, the positiveselection marker and/or negative selection marker comprises an affinitybased screening marker, or a transcriptional modulator protein. In oneembodiment, the same polynucleotide encodes both the positive selectionmarker and the negative selection marker.

In one embodiment, the polynucleotide that encodes the positiveselection marker and/or negative selection marker of the invention cancomprises at least two selector codons, which each or both can compriseat least two different selector codons or at least two of the sameselector codons.

Additional levels of selection/screening stringency can also be used inthe methods of the invention. In one embodiment, the methods cancomprise, e.g., providing a varying amount of an inactive synthetase instep (a), (b) or both (a) and (b), where the varying amount of theinactive synthetase provides an additional level of selection orscreening stringency. In one embodiment, step (a), (b) or both steps (a)and (b) of the method for producing an O-RS includes varying a selectionor screening stringency, e.g., of the positive and/or negative selectionmarker. The method optionally includes subjecting the O-RS thatpreferentially aminoacylates the O-tRNA with the unnatural amino acid toan additional selection round, e.g., an additional positive selectionround(s), an additional negative selection round(s) or combinations ofboth additional positive and negative selection rounds.

In one embodiment, the selecting/screening comprises one or morepositive or negative selection/screening chosen from, e.g., a change inamino acid permeability, a change in translation efficiency, a change intranslational fidelity, etc. The one or more change is based upon amutation in one or more polynucleotide that encodes a component oforthogonal tRNA-tRNA synthetase pair is used to produce protein.

Typically, the library of RSs (e.g., a library of mutant RSs) comprisesRSs derived from at least one aminoacyl-tRNA synthetase (RS), e.g., froma non-eukaryotic organism. In one embodiment, the library of RSs isderived from an inactive RS, e.g., where the inactive RS is generated bymutating an active RS. In another embodiment, the inactive RS comprisesan amino acid binding pocket and one or more amino acids that comprisethe binding pocket are substituted with one or more different aminoacids, e.g., the substituted amino acids are substituted with alanines.

In certain embodiments, the method of producing an O-RS further includesperforming random mutation, site-specific mutation, recombination,chimeric construction, or any combination thereof, on a nucleic acidthat encodes an RS, thereby producing the library of mutant RSs. Incertain embodiments, the method further includes, e.g., (c) isolating anucleic acid that encodes the O-RS; (d) generating from the nucleic acida set of polynucleotides that encode mutated O-RSs (e.g., by randommutagenesis, site-specific mutagenesis, chimeric construction,recombination or any combination thereof); and, (e) repeating steps (a)and/or (b) until a mutated O-RS is obtained that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid. In one aspect ofthe invention, steps (c)-(e) are performed at least two times.

Methods of producing O-tRNA/O-RS pairs are also a feature of theinvention. In one embodiment, the O-RS is obtained as described aboveand the O-tRNA is obtained by subjecting to negative selection apopulation of eukaryotic cells of a first species, where the eukaryoticcells comprise a member of a library of tRNAs, to eliminate cells thatcomprise a member of the library of tRNAs that is aminoacylated by anaminoacyl-tRNA synthetase (RS) that is endogenous to the eukaryoticcells. This provides a pool of tRNAs that are orthogonal to theeukaryotic cell of the first species. In one aspect of the invention,the library of tRNAs comprises tRNAs derived from at least one tRNA,e.g., from a non-eukaryotic organism. In another aspect of theinvention, the library of aminoacyl-tRNA synthetases (RSs) comprises RSsderived from at least one aminoacyl-tRNA synthetase (RS), e.g., from anon-eukaryotic organism. In yet another aspect of the invention, thelibrary of tRNAs comprises tRNAs derived from at least one tRNA from afirst non-eukaryotic organism. The library of aminoacyl-tRNA synthetases(RSs) optionally comprises RSs derived from at least one aminoacyl-tRNAsynthetase (RS) from a second non-eukaryotic organism. In oneembodiment, the first and second non-eukaryotic organisms are the same.Alternatively, the first and second non-eukaryotic organisms can bedifferent. Specific O-tRNA/O-RS pairs produced by the methods of theinvention are also a feature of the invention.

Another feature of the invention is a method for producing translationalcomponents in one species and introducing the selected/screenedtranslational components into a second species. For example, the methodof producing a O-tRNA/O-RS pair in a first species (e.g., a eukaryoticspecies, such as a yeast and the like) further includes introducing anucleic acid that encodes the O-tRNA and a nucleic acid that encodes theO-RS into a eukaryotic cell of a second species (e.g., a mammal, aninsect, a fungus, an algae, a plant and the like). The second speciescan use the introduced translational components to incorporate anunnatural amino acid into a growing polypeptide chain in vivo, e.g.,during translation.

In another example, a method of producing an orthogonal aminoacyl-tRNAsynthetase (O-RS) that preferentially aminoacylates an orthogonal tRNAwith an unnatural amino acid in a eukaryotic cell includes: (a)subjecting to positive selection, in the presence of an unnatural aminoacid, a population of eukaryotic cells of a first species (e.g., aeukaryotic species, such as a yeast or the like). The eukaryotic cellsof the first species each comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker. The cells thatsurvive the positive selection comprise an active RS that aminoacylatesthe orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid.The cells that survive the positive selection are subjected to negativeselection in the absence of the unnatural amino acid to eliminate activeRSs that aminoacylate the O-tRNA with a natural amino acid, therebyproviding an O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. A nucleic acid that encodes the O-tRNA and anucleic acid that encodes the O-RS are introduced into a eukaryotic cellof a second species (e.g., mammal, an insect, a fungus, an algae, aplant and/or the like). These components, when translated in the secondspecies, can be used to incorporate unnatural amino acids into a proteinor polypeptide of interest in the second species. In one embodiment, theO-tRNA and/or the O-RS are introduced into a eukaryotic cell of a secondspecies.

In certain embodiments, the O-tRNA is obtained by subjecting to negativeselection a population of eukaryotic cells of a first species, where theeukaryotic cells comprise a member of a library of tRNAs, to eliminatecells that comprise a member of the library of tRNAs that isaminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous tothe eukaryotic cells. This provides a pool of tRNAs that are orthogonalto the eukaryotic cell of the first species and the second species.

In one aspect, the invention comprises a composition comprising aprotein, wherein the protein comprises at least one unnatural amino acidand at least one post-translational modification, wherein the at leastone post-translational modification comprises attachment of a moleculecomprising a second reactive group by a [3+2] cycloaddition to the atleast one unnatural amino acid comprising a first reactive group.

Thus, proteins (or polypeptides of interest) with at least one unnaturalamino acid are also a feature of the invention. In certain embodimentsof the invention, a protein with at least one unnatural amino acidincludes at least one post-translational modification. In oneembodiment, the at least one post-translational modification comprisesattachment of a molecule (e.g., a dye, a polymer, e.g., a derivative ofpolyethylene glycol, a photocrosslinker, a cytotoxic compound, anaffinity label, a derivative of biotin, a resin, a second protein orpolypeptide, a metal chelator, a cofactor, a fatty acid, a carbohydrate,a polynucleotide (e.g., DNA, RNA, etc.), etc.) comprising a secondreactive group by a [3+2] cycloaddition to the at least one unnaturalamino acid comprising a first reactive group. For example, the firstreactive group is an alkynyl moiety (e.g., in the unnatural amino acidp-propargyloxyphenylalanine) (this group is also sometimes refer to asan acetylene moiety) and the second reactive group is an azido moiety.In another example, the first reactive group is the azido moiety (e.g.,in the unnatural amino acid p-azido-L-phenylalanine) and the secondreactive group is the alkynyl moiety. In certain embodiments, a proteinof the invention includes at least one unnatural amino acid (e.g., aketo unnatural amino acid) comprising at least one post-translationalmodification, where the at least one post-translational modificationcomprises a saccharide moiety. In certain embodiments, thepost-translational modification is made in vivo in a eukaryotic cell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by a eukaryoticcell, where the post-translational modification is not made by aprokaryotic cell. Examples of post-translational modifications include,but are not limited to, acetylation, acylation, lipid-modification,palmitoylation, palmitate addition, phosphorylation, glycolipid-linkagemodification, and the like. In one embodiment, the post-translationalmodification comprises attachment of an oligosaccharide to an asparagineby a GlcNAc-asparagine linkage (e.g., where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (e.g., Gal-GalNAc, Gal-GlcNAc, etc.) to a serine orthreonine by a GalNAc-serine, a GalNAc-threonine, a GlcNAc-serine, or aGlcNAc-threonine linkage. In certain embodiments, a protein orpolypeptide of the invention can comprise a secretion or localizationsequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion,and/or the like.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or even at least 99% ormore identical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and/or thelike), and they comprise one or more unnatural amino acid. In oneembodiment, a composition of the invention includes a protein orpolypeptide of interest and an excipient (e.g., a buffer, apharmaceutically acceptable excipient, etc.).

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different sitesin the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

Examples of a protein (or polypeptide of interest) include, but are notlimited to, e.g., a cytokine, a growth factor, a growth factor receptor,an interferon, an interleukin, an inflammatory molecule, an oncogeneproduct, a peptide hormone, a signal transduction molecule, a steroidhormone receptor, erythropoietin (EPO), insulin, human growth hormone,an Alpha-1 antitrypsin, an Angiostatin, an Antihemolytic factor, anantibody, an Apolipoprotein, an Apoprotein, an Atrial natriureticfactor, an Atrial natriuretic polypeptide, an Atrial peptide, a C—X—Cchemokine, T39765, NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, aGCP-2, an NAP-4, an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, acytokine, a CC chemokine, a Monocyte chemoattractant protein-1, aMonocyte chemoattractant protein-2, a Monocyte chemoattractantprotein-3, a Monocyte inflammatory protein-1 alpha, a Monocyteinflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCCl, T58847,D31065, T64262, a CD40, a CD40 ligand, a C-kit Ligand, a Collagen, aColony stimulating factor (CSF), a Complement factor 5a, a Complementinhibitor, a Complement receptor 1, a cytokine, DHFR, an epithelialNeutrophil Activating Peptide-78, a GROα/MGSA, a GROβ, a GROγ a MIP-1α,a MIP-1δ, a MCP-1, an Epidermal Growth Factor (EGF), an epithelialNeutrophil Activating Peptide, an Erythropoietin (EPO), an Exfoliatingtoxin, a Factor IX, a Factor VII, a Factor VIII, a Factor X, aFibroblast Growth Factor (FGF), a Fibrinogen, a Fibronectin, a G-CSF, aGM-CSF, a Glucocerebrosidase, a Gonadotropin, a growth factor, a growthfactor receptor, a Hedgehog protein, a Hemoglobin, a Hepatocyte GrowthFactor (HGF), a Hirudin, a Human serum albumin, an ICAM-1, an ICAM-1receptor, an LFA-1, an LFA-1 receptor, an Insulin, an Insulin-likeGrowth Factor (IGF), an IGF-I, an an interferon, an IFN-α, an IFN-β, anIFN-γ, an interleukin, an IL-1, an IL-2, an IL-3, an IL-4, an IL-5, anIL-6, an IL-7, an IL-8, an IL-9, an IL-10, an IL-11, an IL-12, aKeratinocyte Growth Factor (KGF), a Lactoferrin, a leukemia inhibitoryfactor, a Luciferase, a Neurturin, a Neutrophil inhibitory factor (NIF),an oncostatin M, an Osteogenic protein, an oncogene product, aParathyroid hormone, a PD-ECSF, a PDGF, a peptide hormone, a HumanGrowth Hormone, a Pleiotropin, a Protein A, a Protein G, a Pyrogenicexotoxins A, B, or C, a Relaxin, a Renin, an SCF, a Soluble complementreceptor I, a Soluble I-CAM 1, a Soluble interleukin receptors, aSoluble TNF receptor, a Somatomedin, a Somatostatin, a Somatotropin, aStreptokinase, a Superantigens, a Staphylococcal enterotoxins, an SEA,an SEB, an SEC1, an SEC2, an SEC3, an SED, an SEE, a steroid hormonereceptor, a Superoxide dismutase (SOD), a Toxic shock syndrome toxin, aThymosin alpha 1, a Tissue plasminogen activator, a tumor growth factor(TGF), a TGF-α, a TGF-β, a Tumor Necrosis Factor, a Tumor NecrosisFactor alpha, a Tumor necrosis factor beta, a Tumor necrosis factorreceptor (TNFR), a VLA-4 protein, a VCAM-1 protein, a VascularEndothelial Growth Factor (VEGEF), a Urokinase, a Mos, a Ras, a Raf, aMet; a p53, a Tat, a Fos, a Myc, a Jun, a Myb, a Rel, an estrogenreceptor, a progesterone receptor, a testosterone receptor, analdosterone receptor, an LDL receptor, a SCF/c-Kit, a CD40L/CD40, aVLA-4/VCAM-1, an ICAM-1/LFA-1, a hyalurin/CD44, a corticosterone, aprotein present in Genebank or other available databases, and the like,and/or a portion thereof. In one embodiment, the polypeptide of interestincludes a transcriptional modulator protein (e.g., a transcriptionalactivator protein (such as GAL4), or a transcriptional repressorprotein, etc.) or a portion thereof.

Compositions of a GAL4 protein, or portion thereof, in a eukaryotic cellare also a feature of the invention. Typically, the GAL4 protein orportion thereof comprises at least one unnatural amino acid.

A eukaryotic cell of the invention provides the ability to synthesizeproteins that comprise unnatural amino acids in large useful quantities.For example, proteins comprising an unnatural amino acid can be producedat a concentration of, e.g., at least 10 μg/liter, at least 50 μg/liter,at least 75 μg/liter, at least 100 μg/liter, at least 200 μg/liter, atleast 250 μg/liter, or at least 500 μg/liter or more of protein in acell extract, a buffer, a pharmaceutically acceptable excipient, and/orthe like. In certain embodiments, a composition of the inventionincludes, e.g., at least 10 μg, at least 50 μg, at least 75 μg, at least100 μg, at least 200 μg, at least 250 μg, or at least 500 μg or more ofprotein that comprises a unnatural amino acid.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) is encoded by a nucleic acid. Typically, the nucleicacid comprises at least one selector codon, at least two selectorcodons, at least three selector codons, at least four selector codons,at least five selector codons, at least six selector codons, at leastseven selector codons, at least eight selector codons, at least nineselector codons, or even ten or more selector codons.

The invention also provides methods for producing, in a eukaryotic cell,at least one protein comprising at least one unnatural amino acid (aswell as proteins produced by such methods). The methods include, e.g.,growing, in an appropriate medium, a eukaryotic cell that comprises anucleic acid that comprises at least one selector codon and encodes theprotein. The eukaryotic cell also comprises an orthogonal tRNA (O-tRNA)that functions in the cell and recognizes the selector codon and anorthogonal aminoacyl tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid, and the mediumcomprises an unnatural amino acid. In one embodiment, the O-RSaminoacylates the O-tRNA with the unnatural amino acid e.g., at least45%, at least 50%, at least 60%, at least 75%, at least 80%, at least90%, at least 95%, or even 99% or more as efficiently as does an O-RShaving an amino acid sequence, e.g., as set forth in SEQ ID NO.: 86 or45. In another embodiment, the O-tRNA comprises, is processed from, oris encoded by SEQ ID NO.: 64 or 65, or a complementary polynucleotidesequence thereof. In yet another embodiment, the O-RS comprises an aminoacid sequence as set forth in any one of SEQ ID NO.: 36-63 (e.g., 36-47,48-63, or any other subset of 36-63), and/or 86.

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (e.g., a dye, a polymer, e.g., a derivative of polyethyleneglycol, a photocrosslinker, a cytotoxic compound, an affinity label, aderivative of biotin, a resin, a second protein or polypeptide, a metalchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide(e.g., DNA, RNA, etc.), etc.) that comprises a second reactive group.The first reactive group reacts with the second reactive group to attachthe molecule to the unnatural amino acid through a [3+2] cycloaddition.In one embodiment, the first reactive group is an alkynyl or azidomoiety and the second reactive group is an azido or alkynyl moiety. Forexample, the first reactive group is the alkynyl moiety (e.g., inunnatural amino acid p-propargyloxyphenylalanine) and the secondreactive group is the azido moiety. In another example, the firstreactive group is the azido moiety (e.g., in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In certain embodiments, the encoded protein comprises a therapeuticprotein, a diagnostic protein, an industrial enzyme, or portion thereof.In one embodiment, the protein that is produced by the method is furthermodified through the unnatural amino acid. For example, the unnaturalamino acid is modified through, e.g., a nucleophilic-electrophilicreaction, through a [3+2] cycloaddition, etc. In another embodiment, theprotein produced by the method is modified by at least onepost-translational modification (e.g., N-glycosylation, O-glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike) in vivo.

Methods of producing a screening or selecting transcriptional modulatorprotein are also provided (as are screening or selecting transcriptionalmodulator proteins produced by such methods). The methods include, e.g.,selecting a first polynucleotide sequence, where the polynucleotidesequence encodes a nucleic acid binding domain; and mutating the firstpolynucleotide sequence to include at least one selector codon. Thisprovides a screening or selecting polynucleotide sequence. The methodsalso include, e.g., selecting a second polynucleotide sequence, wherethe second polynucleotide sequence encodes a transcriptional activationdomain; providing a construct that comprises the screening or selectingpolynucleotide sequence operably linked to the second polynucleotidesequence; and, introducing the construct, an unnatural amino acid, anorthogonal tRNA synthetase (O-RS) and an orthogonal tRNA (O-tRNA), intoa cell. With these components, the O-RS preferentially aminoacylates theO-tRNA with the unnatural amino acid and the O-tRNA recognizes theselector codon and incorporates the unnatural amino acid into thenucleic acid binding domain, in response to the selector codon in thescreening or selecting polynucleotide sequence. This provides thescreening or selecting transcriptional modulator protein.

In certain embodiments, the compositions and the methods of theinvention include eukaryotic cells. A eukaryotic cell of the inventionincludes any of, e.g., a mammalian cell, a yeast cell, a fungus cell, aplant cell, an insect cell, etc. The translation components of theinvention can be derived from a variety of organisms, e.g.,non-eukaryotic organisms, such as a prokaryotic organism (e.g., E. coli,Bacillus stearothermophilus, or the like), or an archaebacterium, ore.g., a eukaryotic organism.

A selector codon of the invention expands the genetic codon framework ofeukaryotic protein biosynthetic machinery. Any of a variety of selectorcodons can be used in the invention, including stop codons (e.g., anamber codon, an ochre codon, or an opal stop codon), nonsense codons,rare codons, four (or more) base codons, and/or the like.

Examples of unnatural amino acids that can be used in the compositionsand methods described herein include (but are not limited to): ap-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, a p-propargyloxyphenylalanine, ap-propargyl-phenylalanine, an L-3-(2-naphthyl)alanine, a3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine,a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine,an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-bromophenylalanine, ap-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnaturalanalogue of a tyrosine amino acid; an unnatural analogue of a glutamineamino acid; an unnatural analogue of a phenylalanine amino acid; anunnatural analogue of a serine amino acid; an unnatural analogue of athreonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo,hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl,seleno, ester, thioacid, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, oramino substituted amino acid, or any combination thereof; an amino acidwith a photoactivatable cross-linker; a spin-labeled amino acid; afluorescent amino acid; a metal binding amino acid; a metal-containingamino acid; a radioactive amino acid; a photocaged and/orphotoisomerizable amino acid; a biotin or biotin-analogue containingamino acid; a keto containing amino acid; an amino acid comprisingpolyethylene glycol or polyether; a heavy atom substituted amino acid; achemically cleavable or photocleavable amino acid; an amino acid with anelongated side chain; an amino acid containing a toxic group; a sugarsubstituted amino acid; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an α-hydroxy containing acid; an amino thioacid; an α,α disubstituted amino acid; a β-amino acid; a cyclic aminoacid other than proline or histidine, an aromatic amino acid other thanphenylalanine, tyrosine or tryptophan, and/or the like.

The invention also provides polypeptides (O-RSs) and polynucleotides,e.g., O-tRNAs, polynucleotides that encode O-RSs or portions thereof(e.g., the active site of the synthetase), oligonucleotides used toconstruct aminoacyl-tRNA synthetase mutants, polynucleotides that encodea protein or polypeptide of interest that comprise one or more selectorcodon, etc. For example, a polypeptide of the invention includes apolypeptide that comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any other subset of36-63), and/or 86, a polypeptide that comprises an amino acid sequenceencoded by a polynucleotide sequence as set forth in any one of SEQ IDNO.: 3-35 (e.g., 3-19, 20-35, or any other subset of sequences 3-35),and a polypeptide that is specifically immunoreactive with an antibodyspecific for a polypeptide that comprises an amino acid sequence asshown in any one of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any othersubset of 36-63), and/or 86, or a polypeptide that comprises an aminoacid sequence encoded by a polynucleotide sequence as shown in any oneof SEQ ID NO.: 3-35 (e.g., 3-19, 20-35, or any other subset of sequences3-35).

Also included among the polypeptides of the invention is a polypeptidethat comprises an amino acid sequence that is at least 90% identical tothat of a naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS)(e.g., SEQ ID NO.:2) and comprises two or more amino acids of groups A-E(noted above). Similarly, polypeptides of the invention also optionallyinclude a polypeptide that comprises at least 20 contiguous amino acidsof any one of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any other subsetof 36-63), and/or 86, and two or more amino acid substitutions asindicated above in groups A-E. An amino acid sequence comprising aconservative variation of any of the above polypeptides is also includedas a polypeptide of the invention.

In one embodiment, a composition includes a polypeptide of the inventionand an excipient (e.g., buffer, water, pharmaceutically acceptableexcipient, etc.). The invention also provides an antibody or antiseraspecifically immunoreactive with a polypeptide of the invention.

Polynucleotides are also provided in the invention. Polynucleotides ofthe invention include those that encode proteins or polypeptides ofinterests of the invention with one or more selector codon. In addition,polynucleotides of the invention include, e.g., a polynucleotidecomprising a nucleotide sequence as set forth in any one of SEQ ID NO.:3-35 (e.g., 3-19, 20-35, or any other subset of sequences 3-35), 64-85;a polynucleotide that is complementary to or that encodes apolynucleotide sequence thereof; and/or a polynucleotide encoding apolypeptide that comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any other subset of36-63), and/or 86, or a conservative variation thereof. A polynucleotideof the invention also includes a polynucleotide that encodes apolypeptide of the invention. Similarly, a nucleic acid that hybridizesto a polynucleotide indicated above under highly stringent conditionsover substantially the entire length of the nucleic acid is apolynucleotide of the invention.

A polynucleotide of the invention also includes a polynucleotide thatencodes a polypeptide that comprises an amino acid sequence that is atleast 90% identical to that of a naturally occurring tyrosylaminoacyl-tRNA synthetase (TyrRS) (e.g., SEQ ID NO.: 2) and comprisestwo or more mutations as indicated above in groups A-E (noted above). Apolynucleotide that is that is at least 70%, (or at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or least99% or more) identical to a polynucleotide indicated above and/or apolynucleotide comprising a conservative variation of any of thepolynucleotides indicated above are also included among thepolynucleotides of the invention.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

In another aspect, the invention provides compositions of compounds andmethods of producing such compounds. For example, compounds include,e.g., an unnatural amino acid (such as p-(propargyloxy)-phenyalanine(e.g., 1 in FIG. 11), azido dyes (such as shown in chemical structure 4and chemical structure 6), an alkynyl polyethylene glycol (e.g., asshown in chemical structure 7), where n is an integer between, e.g., 50and 10,000, 75 and 5,000, 100 and 2,000, 100 and 1,000, etc., and thelike. In embodiment of the invention, the alkynyl polyethylene glycolhas a molecular weight of, e.g., about 5,000 to about 100,000 Da, about20,000 to about 50,000 Da, about 20,000 to about 10,000 Da (e.g., 20,000Da).

Various compositions comprising these compounds, e.g., with proteins andcells, are also provided. In one aspect, the composition that includesthe p-(propargyloxy)-phenyalanine unnatural amino acid, further includesan orthogonal tRNA. The unnatural amino acid can be bonded (e.g.,covalently) to the orthogonal tRNA, e.g., covalently bonded to theorthogonal tRNA though an amino-acyl bond, covalently bonded to a 3′OHor a 2′OH of a terminal ribose sugar of the orthogonal tRNA, etc.

In one aspect of the invention, a protein comprising an azido dye (e.g.,of chemical structure 4 or chemical structure 6), further includes atleast one unnatural amino acid (e.g., an alkynyl amino acid), where theazido dye is attached to the unnatural amino acid through a [3+2]cycloaddition.

In one embodiment, a protein comprises the alkynyl polyethylene glycolof chemical structure 7. In another embodiment, the composition furtherincludes at least one unnatural amino acid (e.g., an azido amino acid),wherein the alkynyl polyethylene glycol is attached to an unnaturalamino acid through a [3+2] cycloaddition.

Methods for synthesizing various compounds are included in theinvention. For example, a method for synthesizing ap-(propargyloxy)phenyalanine compound is provided. For example, themethod comprises (a) suspending N-tert-butoxycarbonyl-tyrosine and K₂CO₃in anhydrous DMF; (b) adding propargyl bromide to the reaction mixtureof (a) and alkylating the hydroxyl and the carboxyl group, resulting inan protected intermediate compound having the structure:

and (c) mixing the protected intermediate compound with anhydrous HCl inMeOH and deprotecting the amine moiety, thereby synthesizing thep-(propargyloxy)phenyalanine compound. In one embodiment, the methodfurther comprises (d) dissolving the p-(propargyloxy)phenylalanine HClin aqueous NaOH and MeOH and stirring it at room temperature; (e)adjusting the pH of to pH 7; and (f) precipitating thep-(propargyloxy)phenylalanine compound.

Methods for synthesizing azido dyes are also provided. For example, amethod comprises: (a) providing a dye compound comprising a sulfonylhalide moiety; (b) warming the dye compound to room temperature in thepresence of 3-azidopropylamine and triethylamine and coupling an aminemoiety of the 3-azidopropylamine to the halide position of the dyecompound, thereby synthesizing the azido dye. In one embodiment, the dyecompound comprises dansyl chloride, and the azido dye comprises thecomposition of chemical structure 4. In one aspect, the method furthercomprises purifying the azido dye from the reaction mixture.

In another example, a method for synthesizing an azido dye comprises (a)providing an amine-containing dye compound; (b) combining theamine-containing dye compound with a carbodiimide and4-(3-azidopropylcarbamoyl)-butyric acid in a suitable solvent, andcoupling a carbonyl group of the acid to the amine moiety of the dyecompound, thereby synthesizing the azido dye. In one embodiment, thecarbodiimine comprises 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDCI). In one aspect, the amine-containing dye comprisesfluoresceinamine, and the suitable solvent comprises pyridine. Forexample, the amine-containing dye comprises fluoresceinamine and theazido dye comprises the composition of chemical structure 6. In oneembodiment, the method further comprises (c) precipitating the azidodye; (d) washing the precipitate with HCl; (e) dissolving the washedprecipitate in EtOAc; and (f) precipitating the azido dye in hexanes.

Methods for synthesizing a propargyl amide polyethylene glycol are alsoprovided. For example, the method comprises reacting propargylamine withpolyethylene glycol (PEG)-hydroxysuccinimide ester in an organic solvent(e.g., CH₂Cl₂) at room temperature, resulting in the propargyl amidepolyethylene glycol of chemical structure 7. In one embodiment, themethod further comprises precipitating the propargylamide polyethyleneglycol using ethyl acetate. In one aspect, the method further includesrecrystallizing the propargylamide polyethylene glycol in methanol; anddrying the product under a vacuum.

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes a container containing apolynucleotide sequence encoding an O-tRNA or an O-tRNA, and apolynucleotide sequence encoding an O-RS or an O-RS. In one embodiment,the kit further includes at least one unnatural amino acid. In anotherembodiment, the kit further comprises instructional materials forproducing the protein.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acell” includes a combination of two or more cells; reference to“bacteria” includes mixtures of bacteria, and the like.

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

Homologous: Proteins and/or protein sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral proteinor protein sequence. Similarly, nucleic acids and/or nucleic acidsequences are homologous when they are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. For example, any naturally occurring nucleic acid can bemodified by any available mutagenesis method to include one or moreselector codon. When expressed, this mutagenized nucleic acid encodes apolypeptide comprising one or more unnatural amino acid. The mutationprocess can, of course, additionally alter one or more standard codon,thereby changing one or more standard amino acid in the resulting mutantprotein, as well. Homology is generally inferred from sequencesimilarity between two or more nucleic acids or proteins (or sequencesthereof). The precise percentage of similarity between sequences that isuseful in establishing homology varies with the nucleic acid and proteinat issue, but as little as 25% sequence similarity is routinely used toestablish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used toestablish homology. Methods for determining sequence similaritypercentages (e.g., BLASTP and BLASTN using default parameters) aredescribed herein and are generally available.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNAsynthetase (O-RS)) that functions with endogenous components of a cellwith reduced efficiency as compared to a corresponding molecule that isendogenous to the cell or translation system, or that fails to functionwith endogenous components of the cell. In the context of tRNAs andaminoacyl-tRNA synthetases, orthogonal refers to an inability or reducedefficiency, e.g., less than 20% efficient, less than 10% efficient, lessthan 5% efficient, or less than 1% efficient, of an orthogonal tRNA tofunction with an endogenous tRNA synthetase compared to an endogenoustRNA to function with the endogenous tRNA synthetase, or of anorthogonal aminoacyl-tRNA synthetase to function with an endogenous tRNAcompared to an endogenous tRNA synthetase to function with theendogenous tRNA. The orthogonal molecule lacks a functional endogenouscomplementary molecule in the cell. For example, an orthogonal tRNA in acell is aminoacylated by any endogenous RS of the cell with reduced oreven zero efficiency, when compared to aminoacylation of an endogenoustRNA by the endogenous RS. In another example, an orthogonal RSaminoacylates any endogenous tRNA in a cell of interest with reduced oreven zero efficiency, as compared to aminoacylation of the endogenoustRNA by an endogenous RS. A second orthogonal molecule can be introducedinto the cell that functions with the first orthogonal molecule. Forexample, an orthogonal tRNA/RS pair includes introduced complementarycomponents that function together in the cell with an efficiency (e.g.,50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80%efficiency, 90% efficiency, 95% efficiency, or 99% or more efficiency)to that of a corresponding tRNA/RS endogenous pair.

Complementary: The term “complementary” refers to components of anorthogonal pair, O-tRNA and O-RS that can function together, e.g., wherethe O-RS aminoacylates the O-tRNA.

Preferentially aminoacylates: The term “preferentially aminoacylates”refers to an efficiency, e.g., 70% efficient, 75% efficient, 85%efficient, 90% efficient, 95% efficient, or 99% or more efficient, atwhich an O-RS aminoacylates an O-tRNA with an unnatural amino acid ascompared to the O-RS aminoacylating a naturally occurring tRNA or astarting material used to generate the O-tRNA. The unnatural amino acidis incorporated into a growing polypeptide chain with high fidelity,e.g., at greater than 75% efficiency for a given selector codon, atgreater than about 80% efficiency for a given selector codon, at greaterthan about 90% efficiency for a given selector codon, at greater thanabout 95% efficiency for a given selector codon, or at greater thanabout 99% or more efficiency for a given selector codon.

Selector codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; rare codons; codons derived fromnatural or unnatural base pairs and/or the like.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system, e.g., by providinga mechanism for incorporating an amino acid into a polypeptide chain inresponse to a selector codon. For example, a suppressor tRNA can readthrough, e.g., a stop codon, a four base codon, a rare codon, and/or thelike.

Recyclable tRNA: The term “recyclable tRNA” refers to a tRNA that isaminoacylated and can be repeatedly reaminoacylated with an amino acid(e.g., an unnatural amino acid) for the incorporation of the amino acid(e.g., the unnatural amino acid) into one or more polypeptide chainsduring translation.

Translation system: The term “translation system” refers to thecollective set of components that incorporate a naturally occurringamino acid into a growing polypeptide chain (protein). Components of atranslation system can include, e.g., ribosomes, tRNAs, synthetases,mRNA, amino acids, and the like. The components of the invention (e.g.,ORS, OtRNAs, unnatural amino acids, etc.) can be added to an in vitro orin vivo translation system, e.g., a eukaryotic cell, e.g., a yeast cell,a mammalian cell, a plant cell, an algae cell, a fungus cell, an insectcell, and/or the like.

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 common naturally occurring aminoacids, seleno cysteine or pyrrolysine.

Derived from: As used herein, the term “derived from” refers to acomponent that is isolated from or made using information from aspecified molecule or organism.

Inactive RS: As used herein, the term “inactive RS” refers to asynthetase that has been mutated so that it no longer can aminoacylateits natural cognate tRNA with an amino acid.

Positive selection or screening marker: As used herein, the term“positive selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, results inidentification of a cell with the positive selection marker from thosewithout the positive selection marker.

Negative selection or screening marker: As used herein, the term“negative selection or screening marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, allows identificationof a cell that does not possess the desired property (e.g., as comparedto a cell that does possess the desired property).

Reporter: As used herein, the term “reporter” refers to a component thatcan be used to select target components of a system of interest. Forexample, a reporter can include a fluorescent screening marker (e.g.,green fluorescent protein), a luminescent marker (e.g., a fireflyluciferase protein), an affinity based screening marker, or selectablemarker genes such as his3, ura3, leu2, lys2, lacZ, β-gal/lacZ(β-galactosidase), Adh (alcohol dehydrogenase), or the like.

Eukaryote: As used herein, the term “eukaryote” refers to organismsbelonging to the phylogenetic domain Eucarya such as animals (e.g.,mammals, insects, reptiles, birds, etc.), ciliates, plants (e.g.,monocots, dicots, algae, etc.), fungi, yeasts, flagellates,microsporidia, protists, etc.

Non-eukaryote: As used herein, the term “non-eukaryote” refers tonon-eukaryotic organisms. For example, a non-eukaryotic organism canbelong to the Eubacteria (e.g., Escherichia coli, Thermus thermophilus,Bacillus stearothermophilus, etc.) phylogenetic domain, or the Archaea(e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

Antibody: The term “antibody,” as used herein, includes, but is notlimited to a polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or fragments thereof, which specifically bindand recognize an analyte (antigen). Examples include polyclonal,monoclonal, chimeric, and single chain antibodies, and the like.Fragments of immunoglobulins, including Fab fragments and fragmentsproduced by an expression library, including phage display, are alsoincluded in the term “antibody” as used herein. See, e.g., Paul,Fundamental Immunology, 4th Ed., 1999, Raven Press, New York, forantibody structure and terminology.

Conservative variant: The term “conservative variant” refers to atranslation component, e.g., a conservative variant O-tRNA or aconservative variant O-RS, that functionally performs like the componentfrom which the conservative variant is based, e.g., an O-tRNA or O-RS,but has variations in the sequence. For example, an O-RS willaminoacylate a complementary O-tRNA or a conservative variant O-tRNAwith an unnatural amino acid, although the O-tRNA and the conservativevariant O-tRNA do not have the same sequence. The conservative variantcan have, e.g., one variation, two variations, three variations, fourvariations, or five or more variations in sequence, as long as theconservative variant is complementary to the corresponding O-tRNA orO-RS.

Selection or screening agent: As used herein, the term “selection orscreening agent” refers to an agent that, when present, allows for aselection/screening of certain components from a population. Forexample, a selection or screening agent includes, but is not limited to,e.g., a nutrient, an antibiotic, a wavelength of light, an antibody, anexpressed polynucleotide (e.g., a transcriptional modulator protein), orthe like. The selection agent can be varied, e.g., by concentration,intensity, etc.

Detectable substance: The term “detectable substance,” as used herein,refers to an agent that, when activated, altered, expressed or the like,allows for the selection/screening of certain components from apopulation. For example, the detectable substance can be a chemicalagent, e.g., 5-fluroorotic acid (5-FOA), which under certain conditions,e.g., expression of a URA3 reporter, becomes detectable, e.g., a toxicproduct that kills cells that express the URA3 reporter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panels A, B and C schematically illustrates a general positiveand negative selection scheme for expanding the genetic code of aeukaryotic cell, e.g., S. cerevisiae. Panel A schematically illustratesthe activated transcription of reporter genes, which are is driven byamber suppression of TAG codons in GAL4. The DNA binding domain isindicated by the striped box and the major and cryptic activationdomains are indicated in the hatched box. Panel B illustrates examplesof reporter genes, e.g., HIS3, LacZ, URA3 in MaV203. Panel Cschematically illustrates plasmids that can be used in the selectionscheme, e.g., pEcTyrRS/tRNA_(CUA) and pGADGAL4xxTAG.

FIG. 2 illustrates the EcTyrRS and tRNA_(CUA) dependent phenotypes of1^(st) generation GAL4 reporters on selective media. DB-AD is a fusionbetween the GAL4 DNA binding domain and activation domain. DB-TAG-AD hasa TAG codon replacing a tyrosine codon in the synthetic linker betweenDB and AD. A5 is an inactive version of EcTyrRS in which 5 residues inthe active site have been mutated to alanine.

FIG. 3, Panel A and B illustrate EcTyrRS and tRNA_(CUA) dependentphenotypes of 2nd generation GAL4 reporters on selective media. The DNAbinding domain is indicated by the striped box and the major and crypticactivation domains are indicated in the hatched box. Panel A illustratesconstructs each with single amino acid mutation in GAL4. Panel Billustrates constructs each with two amino acid mutations in GAL4.

FIG. 4 Panels A, B and C illustrate pGADGAL4 (T44TAG, R110TAG) with andwithout EcTyrRS and various reporters in MaV203. Panel A shows theresults in the presence of X-gal, -Ura, or -Leu, -Trp. Panel B shows theresults in the presence of varying concentrations of 3-AT. Panel C showsthe results in the present of varying percentages of 5-FOA.

FIG. 5 Panels A and B illustrate ONPG hydrolysis with various GAL4mutants, e.g., where residues T44 (A) and R110 (B) are permissive sites.Panel A illustrates the ONPG hydrolysis measurement with various typesof mutations at the T44 site. Panel B illustrates the ONPG hydrolysismeasurement with various types of mutations at the R110 site. ‘GAL4’ isMaV203 transformed with pCL1 and was offscale ˜600 ONPG hydrolysisunits. ‘None’ is MaV203 transformed with plasmids encoding the GAL4 DBand GAL4 AD separately.

FIG. 6 shows the selection of active EcTyrRS clones. MaV203 containing a1:10 mixture of pEcTyrRS-tRNA_(CUA): pA5-tRNA_(CUA) were plated at a 10³dilution on (-Leu, -Trp) plates (left) or (-Leu, -Trp, -His+50 mM 3-AT)plates (right) and processed using XGAL overlay.

FIG. 7, Panels A and B. Panel A illustrates a stereoview of the activesite of B. Stearothermophilus tyrosyl-tRNA synthetase with boundtyrosine. The mutated residues are shown and correspond to residues fromE. coli tyrosyl-tRNA synthetase Tyr³⁷ (B. Stearothermophilus TyrRSresidue Tyr³⁴), Asn¹²⁶ (Asn¹²³), Asp¹⁸² (Asp¹⁷⁶), Phe¹⁸³ (Phe¹⁷⁷), andLeu¹⁸⁶ (Leu¹⁸⁰). Panel B illustrates the structural formulae of examplesof unnatural amino acids (from left to right) p-acetyl-L-phenylalanine(1), p-benzoyl-L-phenylalanine (2), p-azido-L-phenylalanine (3),O-methyl-L-tyrosine (4), and p-iodo-L-tyrosine (5).

FIG. 8, Panels A, B, C and D. Panel A illustrates vectors and reporterconstructs that can be used in selection/screening for orthogonal tRNAs,orthogonal aminoacyl synthetases or pairs of orthogonal tRNA/RS ineukaryotic cells. Panel B illustrates phenotypes of yeast harboring GAL4responsive HIS3, URA3 and lacZ responsive reporters in response toactive (TyrRS) or inactive (A5RS) aminoacyl-tRNA synthetases onselective media. Panel C illustrates an example of a selection schemeused to select mutant synthetases that encode additional amino acids ina eukaryotic cell, e.g., S. cerevisiae, where UAA is an unnatural aminoacid. Panel D illustrates phenotypes of yeast isolated from a selectionwith p-acetyl-L-phenylalanine.

FIG. 9 illustrates protein expression of human superoxide dismutase(hSOD) (33TAG) HIS in S. cerevisiae genetically encoding unnatural aminoacids as indicated in FIG. 7, Panel B.

FIG. 10, Panels A-H illustrate tandem mass spectrum of the trypticpeptide VY*GSIK (SEQ ID NO:87) containing the unnatural amino acids(denoted Y*) as indicated in FIG. 7, Panel B. Panel A illustrates tandemmass spectrum of tryptic peptide with unnatural amino acidp-acetyl-L-phenylalanine (1). Panel B illustrates tandem mass spectrumof tryptic peptide with unnatural amino acid p-benzoyl-L-phenylalanine(2). Panel C illustrates tandem mass spectrum of tryptic peptide withunnatural amino acid p-azido-L-phenylalanine (3). Panel D illustratestandem mass spectrum of tryptic peptide with unnatural amino acidO-methyl-L-tyrosine (4). Panel E illustrates tandem mass spectrum oftryptic peptide with unnatural amino acid p-iodo-L-tyrosine (5). Panel Fillustrates tandem mass spectrum of tryptic peptide with amino acidtrytophan (W) at position Y*. Panel G illustrates tandem mass spectrumof tryptic peptide with amino acid tyrosine (Y) at position Y*. Panel Hillustrates tandem mass spectrum of tryptic peptide with amino acidleucine (L) at position Y*.

FIG. 11 illustrates examples of two unnatural amino acids (1)para-propargyloxyophenylalanine and (2) para-azidophenylalanine.

FIG. 12, Panels A, B and C illustrate SOD expression in the presence orabsence of unnatural amino acids 1 and 2 indicated in FIG. 11. Panel Aillustrates Gelcode Blue stain experiment. Panel B illustrates a westernblot with an anti-SOD antibody. Panel C illustrates a western blot withanti-6×His antibody.

FIG. 13, Panels A, B and C illustrate protein labeling by [3+2]cycyloaddition. Panel A illustrates synthesized dye labels 3-6. Panel Billustrates the reaction between the SOD and the dye. Panel Cillustrates in-gel fluorescence scanning and Gelcode Blue staining.

FIG. 14 illustrates growth of eukaryotic cells, e.g., S. cerevisiaecells, transformed with synthetase mutants in the absence or presence of1 or 2 as indicated in FIG. 11 on SD media lacking uracil.

FIG. 15, Panels A and B, illustrate tandem mass spectrum of the trypticpeptide VY*GSIK (SEQ ID NO:87) containing the azide (Az) (Panel A) oralkyne (Al) (Panel B) unnatural amino acids in position Y* are shownwith their expected fragment ion masses. Arrow indicates observed b(blue) and y (red) ions series for each peptide.

FIG. 16 schematically illustrates the in vivo incorporation of anunnatural amino acid, e.g., para-propargyloxyophenylalanine, into agrowing polypeptide chain and the bioconjugation with small organicmolecules by a [3+2]-cycloaddition reaction through this unnatural aminoacid.

FIG. 17, Panels A, B and C illustrate PEGylation of a protein comprisingan unnatural amino acid using a [3+2] cycloaddition. Panel A illustratesthe reaction of a propargyl amide PEG with a protein comprising an azidoamino acid (e.g., N₃—SOD) in the presence of Cu(I) and phosphate buffer(PB). Panel B illustrates the PEGylation of the protein by gel analysis.Panel C illustrates the synthesis of the propargyl amide PEG.

DETAILED DESCRIPTION

The ability to genetically modify the structures of proteins directly ineukaryotic cells, beyond the chemical constraints imposed by the geneticcode, would provides a powerful molecular tool to both probe andmanipulate cellular processes. The invention provides translationalcomponents that expand the number of genetically encoded amino acids ineukaryotic cells. These include tRNAs (e.g., orthogonal tRNAs(O-tRNAs)), aminoacyl-tRNA synthetases (e.g., orthogonal synthetase(O-RS)), pairs of O-tRNA/O-RSs, and unnatural amino acids.

Typically, O-tRNAs of the invention are expressed and processedefficiently, and function in translation in a eukaryotic cell, but arenot significantly aminoacylated by the host's aminoacyl-tRNAsynthetases. In response to a selector codon, an O-tRNA of the inventiondelivers an unnatural amino acid, which does not encode any of thecommon twenty amino acids, to a growing polypeptide chain during mRNAtranslation.

An O-RS of the invention preferentially aminoacylates an O-tRNA of theinvention with an unnatural amino acid in a eukaryotic cell, but doesnot aminoacylate any of the cytoplasmic host's tRNAs. Moreover, thespecificity of an aminoacyl-tRNA synthetase of the invention providesacceptance of an unnatural amino acid while excluding any endogenousamino acids. Polypeptides that include amino acid sequences of exampleO-RSs, or portions thereof, are also a feature of the invention. Inaddition, polynucleotides that encode translational components, O-tRNAs,O-RSs and portions thereof, are features of the invention.

The invention also provides methods of producing the desiredtranslational components, e.g., O-RS, and or an orthogonal pair(orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase), thatutilizes an unnatural amino acid for use in a eukaryotic cell (andtranslational components produced by such methods). For example, atyrosyl-tRNA synthetase/tRNA_(CUA) pair from E. coli is an O-tRNA/O-RSpair of the invention. In addition, the invention also features methodsof selecting/screening translational components in one eukaryotic cell,and once selected/screened, using those components in a differenteukaryotic cell (a eukaryotic cell that was not used forselection/screening). For example, the selection/screening methods toproduce the translation components for eukaryotic cells can be done inyeast, e.g., Saccharomyces cerevisiae, and then those selectedcomponents can be used in another eukaryotic cell, e.g., another yeastcell, a mammalian cell, an insect cell, a plant cell, a fungus cell,etc.

The invention further provides methods for producing a protein in aeukaryotic cell, where the protein comprises an unnatural amino acid.The protein is produced using the translation components of theinvention. The invention also provides proteins (and proteins producedby the methods of the invention), which include unnatural amino acids.The protein or polypeptide of interest can also include apost-translational modification, e.g., that is added through a [3+2]cycloaddition, or a nucleophilic-electrophilic reaction, that is notmade by a prokaryotic cell, etc. In certain embodiments, methods ofproducing a transcriptional modulator protein with an unnatural aminoacid (and proteins produced by such methods) are also included in theinvention. Compositions, which include proteins that include anunnatural amino acid is also a feature of the invention.

Kits for producing a protein or polypeptide with an unnatural amino acidare also a feature of the invention.

Orthogonal Aminoacyl-tRNA Synthetases (O-RS)

In order to specifically incorporate an unnatural amino acid in to aprotein or polypeptide of interest, in a eukaryotic cell, the substratespecificity of the synthetase is altered so that only the desiredunnatural amino acid, but not any of the common 20 amino acids arecharged to the tRNA. If the orthogonal synthetase is promiscuous, itwill result in mutant proteins with a mixture of natural and unnaturalamino acids at the target position. The invention provides compositionsof, and methods of, producing orthogonal aminoacyl-tRNA synthetases thathave modified substrate specificity for a specific unnatural/amino acid.

A eukaryotic cell that includes an orthogonal aminoacyl-tRNA synthetase(O-RS) is a feature of the invention. The O-RS preferentiallyaminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acidin the eukaryotic cell. In certain embodiments, the O-RS utilizes morethan one unnatural amino acid, e.g., two or more, three or more, etc.Thus, an O-RS of the invention can have the capability to preferentiallyaminoacylate an O-tRNA with different unnatural amino acids. This allowsan additional level of control by selecting which unnatural amino acidor combination of unnatural amino acids are put with the cell and/or byselecting the different amounts of unnatural amino acids that are putwith the cell for their incorporation.

An O-RS of the invention optionally has one or more improved or enhancedenzymatic properties for the unnatural amino acid as compared to anatural amino acid. These properties include, e.g., higher Km, lower Km,higher kcat, lower kcat, lower kcat/km, higher kcat/km, etc., for theunnatural amino acid, as compared to a naturally occurring amino acid,e.g., one of the 20 known common amino acids.

Optionally, the O-RS can be provided to the eukaryotic cell by apolypeptide that includes an O-RS and/or by a polynucleotide thatencodes an O-RS or a portion thereof. For example, an O-RS, or a portionthereof, is encoded by a polynucleotide sequence as set forth in any oneof SEQ ID NO.: 3-35 (e.g., 3-19, 20-35, or any other subset of sequences3-35), or a complementary polynucleotide sequence thereof. In anotherexample, an O-RS comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any other subset of36-63), and/or 86, or a conservative variation thereof. See, e.g.,Tables 5, 6 and 8, and Example 6 herein for sequences of exemplary O-RSmolecules.

An O-RS can also comprise an amino acid sequence that is, e.g., at least90%, at least 95%, at least 98%, at least 99%, or even at least 99.5%identical to that of a naturally occurring tyrosyl aminoacyl-tRNAsynthetase (TyrRS) (e.g., as set forth in SEQ ID NO.:2) and comprisestwo or more amino acids of group A-E. Group A includes valine,isoleucine, leucine, glycine, serine, alanine, or threonine at aposition corresponding to Tyr37 of E. coli TyrRS; group B includesaspartate at a position corresponding to Asn126 of E. coli TyrRS; groupC includes threonine, serine, arginine, asparagine or glycine at aposition corresponding to Asp182 of E. coli TyrRS; group D includesmethionine, alanine, valine, or tyrosine at a position corresponding toPhe183 of E. coli TyrRS; and, group E includes serine, methionine,valine, cysteine, threonine, or alanine at a position corresponding toLeu186 of E. coli TyrRS. Any subset of combinations of these groups area feature of the invention. For example, in one embodiment, the O-RS hastwo or more amino acids selected from valine, isoleucine, leucine, orthreonine occurs at a position corresponding to Tyr37 of E. coli TyrRS;threonine, serine, arginine, or glycine at a position corresponding toAsp182 of E. coli TyrRS; methionine, or tyrosine at a positioncorresponding to Phe183 of E. coli TyrRS; and, serine, or alanine at aposition corresponding to Leu186 of E. coli TyrRS. In anotherembodiment, the O-RS includes two more amino acids selected fromglycine, serine, or alanine at a position corresponding to Tyr37 of E.coli TyrRS, aspartate at a position corresponding to Asn126 of E. coliTyrRS, asparagine at a position corresponding to Asp182 of E. coliTyrRS, alanine, or valine, at a position corresponding to Phe183 of E.coli TyrRS, and/or methionine, valine, cysteine, or threonine, at aposition corresponding to Leu186 of E. coli TyrRS. See also, e.g., Table4, Table 6 and Table 8, herein.

Besides the O-RS, a eukaryotic cell of the invention can includeadditional components, e.g., an unnatural amino acid(s). The eukaryoticcell also includes an orthogonal tRNA (O-tRNA) (e.g., derived from anon-eukaryotic organism, such as Escherichia coli, Bacillusstearothermophilus, and/or the like), where the O-tRNA recognizes aselector codon and is preferentially aminoacylated with the unnaturalamino acid by the O-RS. A nucleic acid that comprises a polynucleotidethat encodes a polypeptide of interest, wherein the polynucleotidecomprises a selector codon that is recognized by the O-tRNA, or acombination of one or more of these, can also be present in the cell.

In one aspect, the O-tRNA mediates the incorporation of the unnaturalamino acid into a protein with, e.g., at least 45%, at least 50%, atleast 60%, at least 75%, at least 80%, at least 90%, at least 95%, or99% or the efficiency of as a tRNA that comprises or is processed from apolynucleotide sequence as set forth in SEQ ID NO.: 65. In anotheraspect, the O-tRNA comprises SEQ ID NO.:65, and the O-RS comprises apolypeptide sequence set forth in any one of SEQ ID NO.: 36-63 (e.g.,36-47, 48-63, or any other subset of 36-63), and/or 86, and/or aconservative variation thereof. See also, e.g., Table 5 and Example 6,herein, for sequences of exemplary O-RS and O-tRNA molecules.

In one example, a eukaryotic cell comprises an orthogonal aminoacyl-tRNAsynthetase (O-RS), an orthogonal tRNA (O-tRNA), an unnatural amino acid,and a nucleic acid that comprises a polynucleotide that encodes apolypeptide of interest, which polynucleotide comprises a selector codonthat is recognized by the O-tRNA. The O-RS preferentially aminoacylatesthe orthogonal tRNA (O-tRNA) with the unnatural amino acid in theeukaryotic cell, and the cell produces the polypeptide of interest inthe absence of the unnatural amino acid with a yield that is, e.g., lessthan 30%, less than 20%, less than 15%, less than 10%, less than 5%,less than 2.5%, etc., of the yield of the polypeptide in the presence ofthe unnatural amino acid.

Methods for producing an O-RS, which are a feature of the invention,optionally include generating a pool of mutant synthetases from theframework of a wild-type synthetase, and then selecting for mutated RSsbased on their specificity for an unnatural amino acid relative to thecommon twenty amino acids. To isolate such a synthetase, the selectionmethods of the are: (i) sensitive, as the activity of desiredsynthetases from the initial rounds can be low and the population small;(ii) “tunable”, since it is desirable to vary the selection stringencyat different selection rounds; and, (iii) general, so that the methodscan be used for different unnatural amino acids.

Methods of producing an orthogonal aminoacyl-tRNA synthetase (O-RS) thatpreferentially aminoacylates an orthogonal tRNA with an unnatural aminoacid in a eukaryotic cell typically include applying a combination of apositive selection followed by a negative selection. In the positiveselection, suppression of the selector codon introduced at nonessentialposition(s) of a positive marker allows the eukaryotic cells to surviveunder positive selection pressure. In the presence of unnatural aminoacids, survivors thus encode active synthetases charging the orthogonalsuppressor tRNA with an unnatural amino acid. In the negative selection,suppression of a selector codon introduced at nonessential position(s)of a negative marker removes synthetases with natural amino acidspecificities. Survivors of the negative and positive selection encodesynthetases that aminoacylate (charge) the orthogonal suppressor tRNAwith unnatural amino acids only (or at least preferentially).

For example, the method includes: (a) subjecting to positive selection,in the presence of an unnatural amino acid, a population of eukaryoticcells of a first species, where the eukaryotic cells each comprise: i) amember of a library of aminoacyl-tRNA synthetases (RSs), ii) anorthogonal tRNA (O-tRNA), iii) a polynucleotide that encodes a positiveselection marker, and iv) a polynucleotide that encodes a negativeselection marker; wherein cells that survive the positive selectioncomprise an active RS that aminoacylates the orthogonal tRNA (O-tRNA) inthe presence of an unnatural amino acid; and, (b) subjecting the cellsthat survive the positive selection to negative selection in the absenceof the unnatural amino acid to eliminate active RSs that aminoacylatethe O-tRNA with a natural amino acid, thereby providing the O-RS thatpreferentially aminoacylates the O-tRNA with the unnatural amino acid.

The positive selection marker can be any of a variety of molecules. Inone embodiment, the positive selection marker is a product that providesa nutritional supplement for growth and the selection is performed on amedium that lacks the nutritional supplement. Examples ofpolynucleotides that encode positive selection markers include, but arenot limited to, e.g., a reporter gene based on complementing the aminoacid auxotrophy of a cell, a his3 gene (e.g., where the his3 geneencodes an imidazole glycerol phosphate dehydratase, detected byproviding 3-aminotriazole (3-AT)), ura3 gene, leu2 gene, lys2 gene, lacZgene, adh gene, etc. See, e.g., G. M. Kishore, & D. M. Shah, (1988),Amino acid biosynthesis inhibitors as herbicides, Annual Review ofBiochemistry 57:627-663. In one embodiment, lacZ production is detectedby ortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis. See, e.g.,I. G. Serebriiskii, & E. A. Golemis, (2000), Uses of lacZ to study genefunction: evaluation of beta-galactosidase assays employed in the yeasttwo-hybrid system, Analytical Biochemistry 285:1-15. Additional positiveselection markers include, e.g., luciferase, green fluorescent protein(GFP), YFP, EGFP, RFP, the product of an antibiotic resistant gene(e.g., chloramphenicol acetyltransferase (CAT)), a transcriptionalmodulator protein (e.g., GAL4), etc. Optionally, a polynucleotide thatencodes a positive selection marker comprises a selector codon.

A polynucleotide that encodes the positive selection marker can beoperably linked to a response element. An additional polynucleotide thatencodes a transcriptional modulator protein that modulates transcriptionfrom the response element, and comprises at least one selector codon,can also be present. The incorporation of the unnatural amino acid intothe transcriptional modulator protein by the O-tRNA aminoacylated withthe unnatural amino acid results in transcription of the polynucleotide(e.g., reporter gene) encoding the positive selection marker. Forexample, see FIG. 1A. Optionally, the selector codon is located in orsubstantially near a portion of the polynucleotide that encodes a DNAbinding domain of the transcriptional modulator protein.

A polynucleotide that encodes the negative selection marker can also beoperably linked to a response element from which transcription ismediated by the transcriptional modulator protein. See, e.g., A. J.DeMaggio, et al., (2000), The yeast split-hybrid system, Method Enzymol.328:128-137; H. M. Shih, et al., (1996), A positive genetic selectionfor disrupting protein-protein interactions: identification of CREBmutations that prevent association with the coactivator CBP, Proc. Natl.Acad. Sci. U.S.A. 93:13896-13901; M. Vidal, et al., (1996), Geneticcharacterization of a mammalian protein-protein interaction domain byusing a yeast reverse two-hybrid system.[comment], Proc. Natl. Acad.Sci. U.S.A. 93:10321-10326; and, M. Vidal, et al., (1996), Reversetwo-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions.[comment], Proc. Natl.Acad. Sci. U.S.A. 93:10315-10320. The incorporation of a natural aminoacid into the transcriptional modulator protein by the O-tRNAaminoacylated with a natural amino acid results in transcription of thenegative selection marker. Optionally, the negative selection markercomprises a selector codon. In one embodiment, the positive selectionmarker and/or negative selection marker of the invention can comprise atleast two selector codons, which each or both can comprise at least twodifferent selector codons or at least two of the same selector codons.

The transcriptional modulator protein is a molecule that binds (directlyor indirectly) to a nucleic acid sequence (e.g., a response element) andmodulates transcription of a sequence that is operably linked to theresponse element. A transcriptional modulator protein can be atranscriptional activator protein (e.g., GAL4, nuclear hormonereceptors, AP1, CREB, LEF/tcf family members, SMADs, VP16, SP1, etc.), atranscriptional repressor protein (e.g., nuclear hormone receptors,Groucho/tle family, Engrailed family, etc), or a protein that can haveboth activities depending on the environment (e.g., LEF/tcf, homoboxproteins, etc.). A response element is typically a nucleic acid sequencethat is recognized by the transcriptional modulator protein or anadditional agent that acts in concert with the transcriptional modulatorprotein.

Another example of a transcriptional modulator protein is thetranscriptional activator protein, GAL4 (see e.g., FIG. 1A). See, e.g.,A. Laughon, et al., (1984), Identification of two proteins encoded bythe Saccharomyces cerevisiae GAL4 gene, Molecular & Cellular Biology4:268-275; A. Laughon, & R. F. Gesteland, (1984), Primary structure ofthe Saccharomyces cerevisiae GAL4 gene, Molecular & Cellular Biology4:260-267; L. Keegan, et al., (1986), Separation of DNA binding from thetranscription-activating function of a eukaryotic regulatory protein,Science 231:699-704; and, M. Ptashne, (1988), How eukaryotictranscriptional activators work, Nature 335:683-689. The N-terminal 147amino acids of this 881 amino acid protein form a DNA binding domain(DBD) that binds DNA sequence specifically. See, e.g., M. Carey, et al.,(1989), An amino-terminal fragment of GAL4 binds DNA as a dimer, J. Mol.Biol. 209:423-432; and, E. Giniger, et al., (1985), Specific DNA bindingof GAL4, a positive regulatory protein of yeast, Cell 40:767-774. TheDBD is linked, by an intervening protein sequence, to a C-terminal 113amino acid activation domain (AD) that can activate transcription whenbound to DNA. See, e.g., J. Ma, & M. Ptashne, (1987), Deletion analysisof GAL4 defines two transcriptional activating segments, Cell48:847-853: and, J. Ma, & M. Ptashne, (1987), The carboxy-terminal 30amino acids of GAL4 are recognized by GAL80, Cell 50:137-142. By placingamber codons towards, e.g., the N-terminal DBD of a single polypeptidethat contains both the N-terminal DBD of GAL4 and its C-terminal AD,amber suppression by the O-tRNA/O-RS pair can be linked totranscriptional activation by GAL4 (FIG. 1, Panel A). GAL4 activatedreporter genes can be used to perform both positive and negativeselections with the gene (FIG. 1, Panel B).

The medium used for negative selection can comprise a selecting orscreening agent that is converted to a detectable substance by thenegative selection marker. In one aspect of the invention, thedetectable substance is a toxic substance. A polynucleotide that encodesa negative selection marker can be, e.g., an ura3 gene. For example, theURA3 reporter can be placed under control of a promoter that containsGAL4 DNA binding sites. When the negative selection marker is produced,e.g., by translation of a polynucleotide encoding the GAL4 with selectorcodons, GAL4 activates transcription of URA3. The negative selection isaccomplished on a medium that comprises 5-fluoroorotic acid (5-FOA),which is converted into a detectable substance (e.g., a toxic substancewhich kills the cell) by the gene product of the ura3 gene. See, e.g.,J. D. Boeke, et al., (1984), A positive selection for mutants lackingorotidine-5′-phosphate decarboxylase activity in yeast: 5-fluorooroticacid resistance, Molecular & General Genetics 197:345-346); M. Vidal, etal., (1996), Genetic characterization of a mammalian protein-proteininteraction domain by using a yeast reverse two-hybrid system.[comment],Proc. Natl. Acad. Sci. U.S.A. 93:10321-10326; and, M. Vidal, et al.,(1996), Reverse two-hybrid and one-hybrid systems to detect dissociationof protein-protein and DNA-protein interactions.[comment], Proc. Natl.Acad. Sci. U.S.A. 93:10315-10320. See also, FIG. 8C.

As with the positive selection marker, the negative selection marker canalso be any of a variety of molecules. In one embodiment, the positiveselection marker and/or the negative selection marker is a polypeptidethat fluoresces or catalyzes a luminescent reaction in the presence of asuitable reactant. For example, negative selection markers include, butare not limited to, e.g., luciferase, green fluorescent protein (GFP),YFP, EGFP, RFP, the product of an antibiotic resistant gene (e.g.,chloramphenicol acetyltransferase (CAT)), the product of a lacZ gene,transcriptional modulator protein, etc. In one aspect of the invention,the positive selection marker and/or the negative selection marker isdetected by fluorescence-activated cell sorting (FACS) or byluminescence. In another example, the positive selection marker and/ornegative selection marker comprise an affinity based screening marker.The same polynucleotide can encode both the positive selection markerand the negative selection marker.

Additional levels of selection/screening stringency can also be used inthe methods of the invention. The selection or screening stringency canbe varied on one or both steps of the method to produce an O-RS. Thiscould include, e.g., varying the amount of response elements in apolynucleotide that encodes the positive and/or negative selectionmarker, adding a varying amount of an inactive synthetase to one or bothof the steps, varying the amount of selection/screening agent that isused, etc. Additional rounds of positive and/or negative selections canalso be performed.

Selecting or screening can also comprise one or more positive ornegative selection or screening that includes, e.g., a change in aminoacid permeability, a change in translation efficiency, a change intranslational fidelity, etc. Typically, the one or more change is basedupon a mutation in one or more polynucleotides that comprise or encodecomponents of an orthogonal tRNA-tRNA synthetase pair that are used toproduce protein.

Model enrichment studies can also be used to rapidly select an activesynthetase from an excess of inactive synthetases. Positive and/ornegative model selection studies can be done. For example, eukaryoticcells that comprise potential active aminoacyl-tRNA synthetases aremixed with a varying fold excess of inactive aminoacyl-tRNA synthetases.A ratio comparison is made between cells grown in a nonselective mediaand assayed by, e.g., X-GAL overlay, and those grown and able to survivein a selective media (e.g., in the absence of histidine and/or uracil)and assayed by, e.g., an X-GAL assay. For a negative model selection,potential active aminoacyl-tRNA synthetases are mixed with a varyingfold excess of inactive aminoacyl-tRNA synthetases and selection isperformed with a negative selection substance, e.g., 5-FOA.

Typically, the library of RSs (e.g., a library of mutant RSs) comprisesRSs derived from at least one aminoacyl-tRNA synthetase (RS), e.g., froma non-eukaryotic organism. In one embodiment, the library of RSs isderived from an inactive RS, e.g., where the inactive RS is generated bymutating an active RS, e.g., at the active site in the synthetase, atthe editing mechanism site in the synthetase, at different sites bycombining different domains of synthetases, or the like. For example,residues in the active site of the RS are mutated to, e.g., alanineresidues. The polynucleotide that encodes the alanine mutated RS is usedas a template to mutagenize the alanine residues to all 20 amino acids.The library of mutant RSs is selected/screened to produce the O-RS. Inanother embodiment, the inactive RS comprises an amino acid bindingpocket and one or more amino acids that comprise the binding pocket aresubstituted with one or more different amino acids. In one example, thesubstituted amino acids are substituted with alanines. Optionally, thepolynucleotide that encodes the alanine mutated RS is used as a templateto mutagenize the alanine residues to all 20 amino acids andscreened/selected.

The method of producing an O-RS can further include producing thelibrary of RSs by using various mutagenesis techniques known in the art.For example, the mutant RSs can be generated by site-specific mutations,random point mutations, homologous recombination, DNA shuffling or otherrecursive mutagenesis methods, chimeric construction or any combinationthereof. For example, a library of mutant RSs can be produced from twoor more other, e.g., smaller, less diverse “sub-libraries.” Once thesynthetases are subject to the positive and negative selection/screeningstrategy, these synthetases can then be subjected to furthermutagenesis. For example, a nucleic acid that encodes the O-RS can beisolated; a set of polynucleotides that encode mutated O-RSs (e.g., byrandom mutagenesis, site-specific mutagenesis, recombination or anycombination thereof) can be generated from the nucleic acid; and, theseindividual steps or a combination of these steps can be repeated until amutated O-RS is obtained that preferentially aminoacylates the O-tRNAwith the unnatural amino acid. In one aspect of the invention, the stepsare performed at least two times.

Additional details for producing O-RS can be found in WO 2002/086075entitled “Methods and compositions for the production of orthogonaltRNA-aminoacyltRNA synthetase pairs.” See also, Hamano-Takaku et al.,(2000) A mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes theUnnatural Amino Acid Azatyrosine More Efficiently than Tyrosine, Journalof Biological Chemistry, 275(51):40324-40328; Kiga et al. (2002), Anengineered Escherichia coli tyrosyl-tRNA synthetase for site-specificincorporation of an unnatural amino acid into proteins in eukaryotictranslation and its application in a wheat germ cell-free system, PNAS99(15): 9715-9723; and, Francklyn et al., (2002), Aminoacyl-tRNAsynthetases: Versatile players in the changing theater of translation;RNA, 8:1363-1372.

Orthogonal tRNAs

Eukaryotic cells that include an orthogonal tRNA (O-tRNA) are providedby the invention. The orthogonal tRNA mediates incorporation of anunnatural amino acid into a protein that is encoded by a polynucleotidethat comprises a selector codon that is recognized by the O-tRNA, invivo. In certain embodiments, an O-tRNA of the invention mediates theincorporation of an unnatural amino acid into a protein with, e.g., atleast 40%, at least 45%, at least 50%, at least 60%, at least 75%, atleast 80%, or even 90% or more as efficiently as tRNA that comprises oris processed in a cell from a polynucleotide sequence as set forth inSEQ ID NO.: 65. See, Table 5, herein.

An example of an O-tRNA of the invention is SEQ ID NO.: 65. (See Example6 and Table 5, herein). SEQ ID NO.: 65 is a pre-splicing/processingtranscript that is optionally processed in the cell, e.g., using thecell's endogenous splicing and processing machinery, and modified toform an active O-tRNA. Typically, a population of such pre-splicingtranscripts form a population of active tRNAs in the cell (the activetRNAs can be in one or more active forms). The invention also includesconservative variations of the O-tRNA and its processed cellularproducts. For example, conservative variations of O-tRNA include thosemolecules that function like the O-tRNA of SEQ ID NO.:65 and maintainthe tRNA L-shaped structure, e.g., in processed form, but do not havethe same sequence (and are other than wild type tRNA molecules).Typically, an O-tRNA of the invention is a recyclable O-tRNA, becausethe O-tRNA can be reaminoacylated in vivo to again mediate theincorporation of the unnatural amino acid into a protein that is encodedby a polynucleotide in response to a selector codon.

The transcription of the tRNA in eukaryotes, but not in prokaryotes, iscarried out by RNA Polymerase III, which places restrictions on theprimary sequence of the tRNA structural genes that can be transcribed ineukaryotic cells. In addition, in eukaryotic cells, tRNAs need to beexported from the nucleus, where they are transcribed, to the cytoplasm,to function in translation. Nucleic acids that encode an O-tRNA of theinvention or a complementary polynucleotide thereof are also a featureof the invention. In one aspect of the invention, a nucleic acid thatencodes an O-tRNA of the invention includes an internal promotersequence, e.g., an A box (e.g., TRGCNNAGY) and a B box (e.g.,GGTTCGANTCC, SEQ ID NO:88). The O-tRNA of the invention can also bepost-transcriptionally modified. For example, post-transcriptionalmodification of tRNA genes in eukaryotes include removal of the 5′- and3′-flanking sequences by Rnase P and a 3′-endonuclease, respectively.The addition of a 3′-CCA sequence is also a post-transcriptionalmodification of a tRNA gene in eukaryotes.

In one embodiment, an O-tRNA is obtained by subjecting to negativeselection a population of eukaryotic cells of a first species, where theeukaryotic cells comprise a member of a library of tRNAs. The negativeselection eliminates cells that comprise a member of the library oftRNAs that is aminoacylated by an aminoacyl-tRNA synthetase (RS) that isendogenous to the eukaryotic cells. This provides a pool of tRNAs thatare orthogonal to the eukaryotic cell of the first species.

Alternatively, or in combination with others methods described above toincorporate an unnatural amino acid into a polypeptide, atrans-translation system can be used. This system involves a moleculecalled tmRNA present in Escherichia coli. This RNA molecule isstructurally related to an alanyl tRNA and is aminoacylated by thealanyl synthetase. The difference between tmRNA and tRNA is that theanticodon loop is replaced with a special large sequence. This sequenceallows the ribosome to resume translation on sequences that have stalledusing an open reading frame encoded within the tmRNA as template. In theinvention, an orthogonal tmRNA can be generated that is preferentiallyaminoacylated with an orthogonal synthetase and loaded with an unnaturalamino acid. By transcribing a gene by the system, the ribosome stalls ata specific site; the unnatural amino acid is introduced at that site,and translation resumes using the sequence encoded within the orthogonaltmRNA.

Additional methods for producing a recombinant orthogonal tRNAs can befound, e.g., in International patent applications WO 2002/086075,entitled “Methods and compositions for the production of orthogonaltRNA-aminoacyltRNA synthetase pairs.” See also, Forster et al., (2003)Programming peptidomimetic synthetases by translating genetic codesdesigned de novo PNAS 100(11):6353-6357; and, Feng et al., (2003),Expanding tRNA recognition of a tRNA synthetase by a single amino acidchange, PNAS 100(10): 5676-5681.

Orthogonal tRNA and Orthogonal Aminoacyl-tRNA Synthetase Pairs

An orthogonal pair is composed of an O-tRNA, e.g., a suppressor tRNA, aframeshift tRNA, or the like, and an O-RS. The O-tRNA is not acylated byendogenous synthetases and is capable of mediating incorporation of anunnatural amino acid into a protein that is encoded by a polynucleotidethat comprises a selector codon that is recognized by the O-tRNA invivo. The O-RS recognizes the O-tRNA and preferentially aminoacylatesthe O-tRNA with an unnatural amino acid in a eukaryotic cell. Methodsfor producing orthogonal pairs along with orthogonal pairs produced bysuch methods and compositions of orthogonal pairs for use in eukaryoticcells are included in the invention. The development of multipleorthogonal tRNA/synthetase pairs can allow the simultaneousincorporation of multiple unnatural amino acids using different codonsin a eukaryotic cell.

An orthogonal O-tRNA/O-RS pair in a eukaryotic cell can be produced byimporting a pair, e.g., a nonsense suppressor pair, from a differentorganism with inefficient cross species aminoacylation. The O-tRNA andO-RS are efficiently expressed and processed in the eukaryotic cell andthe O-tRNA is efficiently exported from the nucleus to the cytoplasm.For example, one such pair is the tyrosyl-tRNA synthetase/tRNA_(CUA)pair from E. coli (see, e.g., H. M. Goodman, et al., (1968), Nature217:1019-24; and, D. G. Barker, et al., (1982), FEBS Letters150:419-23). E. coli tyrosyl-tRNA synthetase efficiently aminoacylatesits cognate E. coli tRNA_(CUA) when both are expressed in the cytoplasmof S. cerevisiae, but does not aminoacylate S. cerevisiae tRNAs. See,e.g., H. Edwards, & P. Schimmel, (1990), Molecular & Cellular Biology10:1633-41; and, H. Edwards, et al., (1991), PNAS United States ofAmerica 88:1153-6. In addition, E. coli tyrosyl tRNA_(CUA) is a poorsubstrate for S. cerevisiae aminoacyl-tRNA synthetases (see, e.g., V.Trezeguet, et al., (1991), Molecular & Cellular Biology 11:2744-51), butfunctions efficiently in protein translation in S. cerevisiae. See,e.g., H. Edwards, & P. Schimmel, (1990) Molecular & Cellular Biology10:1633-41; H. Edwards, et al., (1991), PNAS United States of America88:1153-6; and, V. Trezeguet, et al., (1991), Molecular & CellularBiology 11:2744-51. Moreover, E. coli TyrRS does not have an editingmechanism to proofread an unnatural amino acid ligated to the tRNA.

The O-tRNA and O-RS can be naturally occurring or can be derived bymutation of a naturally occurring tRNA and/or RS, which generateslibraries of tRNAs and/or libraries of RSs, from a variety of organism.See the section entitled “Sources and Hosts” herein. In variousembodiments, the O-tRNA and O-RS are derived from at least one organism.In another embodiment, the O-tRNA is derived from a naturally occurringor mutated naturally occurring tRNA from a first organism and the O-RSis derived from naturally occurring or mutated naturally occurring RSfrom a second organism. In one embodiment, the first and secondnon-eukaryotic organisms are the same. Alternatively, the first andsecond non-eukaryotic organisms can be different.

See sections herein entitled “Orthogonal aminoacyl-tRNA synthetases” and“O-tRNA” for methods of producing O-RSs and O-tRNAs. See also,International patent application WO 2002/086075, entitled “Methods andcompositions for the production of orthogonal tRNA-aminoacyltRNAsynthetase pairs.”

Fidelity, Efficiency, and Yield

Fidelity refers to the accuracy with which a desired molecule, e.g., anunnatural amino acid or amino acid, is incorporated into a growingpolypeptide at a desired position. The translational components of theinvention incorporate unnatural amino acids, with high fidelity, intoproteins in response to a selector codon. For example, using thecomponents of the invention, the efficiency of incorporation of adesired unnatural amino acid into a growing polypeptide chain at adesired position (e.g., in response to a selector codon) is, e.g.,greater than 75%, greater than 85%, greater than 95%, or even greaterthan 99% or more as efficient as compared to unwanted incorporation aspecific natural amino acid being incorporated into the growingpolypeptide chain the desired position.

Efficiency can also refer to the degree with which the O-RSaminoacylates the O-tRNA with the unnatural amino acid as compared to arelevant control. O-RSs of the invention can be defined by theirefficiency. In certain embodiments of the invention, an O-RS is comparedto another O-RS. For example, a O-RS of the invention aminoacylates aO-tRNA with an unnatural amino acid, e.g., at least 40%, at least 50%,at least 60%, at least 75%, at least 80%, at least 90%, at least 95%, oreven 99% or more as efficiently as an O-RS having an amino acidsequence, e.g., as set forth in SEQ ID NO.: 86 or 45) or anotherspecific RS in Table 5) aminoacylates an O-tRNA. In another embodiment,an O-RS of the invention aminoacylates the O-tRNA with the unnaturalamino acid at least 10-fold, at least 20-fold, at least 30-fold, etc.,more efficiently than the O-RS aminoacylates the O-tRNA with a naturalamino acid.

Using the translational components of the invention, the yield of thepolypeptide of interest comprising the unnatural amino acid is, e.g., atleast 5%, at least 10%, at least 20%, at least 30%, at least 40%, 50% ormore, of that obtained for the naturally occurring polypeptide ofinterest from a cell in which the polynucleotide lacks the selectorcodon. In another aspect, the cell produces the polypeptide of interestin the absence of the unnatural amino acid with a yield that is, e.g.,less than 30%, less than 20%, less than 15%, less than 10%, less than5%, less than 2.5%, etc., of the yield of the polypeptide in thepresence of the unnatural amino acid.

Source and Host Organisms

The orthogonal translational components of the invention are typicallyderived from non-eukaryotic organisms for use in eukaryotic cells ortranslation systems. For example, the orthogonal O-tRNA can be derivedfrom a non-eukaryotic organism, e.g., a eubacterium, such as Escherichiacoli, Thermus thermophilus, Bacillus stearothermphilus, or the like, oran archaebacterium, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium such as Haloferax volcanii andHalobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcusfuriosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, whilethe orthogonal O-RS can be derived from a non-eukaryotic organism, e.g.,a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillusstearothermphilus, or the like, or an archaebacterium, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcushorikoshii, Aeuropyrum pernix, or the like. Alternately, eukaryoticsources can also be used, e.g., plants, algae, protists, fungi, yeasts,animals (e.g., mammals, insects, arthropods, etc.), or the like, e.g.,where the components are orthogonal to a cell or translation system ofinterest, or where they are modified (e.g., mutated) to be orthogonal tothe cell or translation system.

The individual components of an O-tRNA/O-RS pair can be derived from thesame organism or different organisms. In one embodiment, the O-tRNA/O-RSpair is from the same organism. For example, the O-tRNA/O-RS pair can bederived from a tyrosyl-tRNA synthetase/tRNA_(CUA) pair from E. coli.Alternatively, the O-tRNA and the O-RS of the O-tRNA/O-RS pair areoptionally from different organisms.

The orthogonal O-tRNA, O-RS or O-tRNA/O-RS pair can be selected orscreened and/or used in a eukaryotic cell to produce a polypeptide withan unnatural amino acid. A eukaryotic cell can be from an of a varietyof sources, e.g., a plant (e.g., complex plant such as monocots, ordicots), an algae, a protist, a fungus, a yeast (e.g., Saccharomycescerevisiae), an animal (e.g., a mammal, an insect, an arthropod, etc.),or the like. Compositions of eukaryotic cells with translationalcomponents of the invention are also a feature of the invention.

The invention also provides for the efficient screening in one speciesfor optional use in that species and/or a second species (optionally,without additional selection/screening). For example, the components ofthe O-tRNA/O-RS are selected or screened in one species, e.g., an easilymanipulated species (such as a yeast cell, etc.) and introduced into asecond eukaryotic species, e.g., a plant (e.g., complex plant such asmonocots, or dicots), an algae, a protist, a fungus, a yeast, an animal(e.g., a mammal, an insect, an arthropod, etc.), or the like, for use inthe in vivo incorporation of an unnatural amino acid in the secondspecies.

For example, Saccharomyces cerevisiae (S. cerevisiae) can be chosen asthe eukaryotic first species, as it is unicellular, has a rapidgeneration time, and relatively well-characterized genetics. See, e.g.,D. Burke, et al., (2000) Methods in Yeast Genetics. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Moreover, since thetranslational machinery of eukaryotes is highly conserved (see, e.g.,(1996) Translational Control. Cold Spring Harbor Laboratory, Cold SpringHarbor, NY; Y. Kwok, & J. T. Wong, (1980), Evolutionary relationshipbetween Halobacterium cutirubrum and eukaryotes determined by use ofaminoacyl-tRNA synthetases as phylogenetic probes, Canadian Journal ofBiochemistry 58:213-218; and, (2001) The Ribosome. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), aaRSs genes for theincorporation of unnatural amino acids discovered in S. cerevisiae canbe introduced into higher eukaryotic organisms and used, in partnershipwith cognate tRNAs (see, e.g., K. Sakamoto, et al., (2002) Site-specificincorporation of an unnatural amino acid into proteins in mammaliancells, Nucleic Acids Res. 30:4692-4699; and, C. Kohrer, et al., (2001),Import of amber and ochre suppressor tRNAs into mammalian cells: ageneral approach to site-specific insertion of amino acid analogues intoproteins, Proc. Natl. Acad. Sci. U.S.A. 98:14310-14315) to incorporateunnatural amino acids.

In one example, the method of producing O-tRNA/O-RS in a first speciesas described herein further includes introducing a nucleic acid thatencodes the O-tRNA and a nucleic acid that encodes the O-RS into aeukaryotic cell of a second species (e.g., a mammal, an insect, afungus, an algae, a plant and the like). In another example, a method ofproducing an orthogonal aminoacyl-tRNA synthetase (O-RS) thatpreferentially aminoacylates an orthogonal tRNA with an unnatural aminoacid in a eukaryotic cell includes: (a) subjecting to positiveselection, in the presence of an unnatural amino acid, a population ofeukaryotic cells of a first species (e.g., yeast and the like). Each ofthe eukaryotic cells comprise: i) a member of a library ofaminoacyl-tRNA synthetases (RSs), ii) an orthogonal tRNA (O-tRNA), iii)a polynucleotide that encodes a positive selection marker, and iv) apolynucleotide that encodes a negative selection marker. The cells thatsurvive the positive selection comprise an active RS that aminoacylatesthe orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid.The cells that survive the positive selection are subjected to negativeselection in the absence of the unnatural amino acid to eliminate activeRSs that aminoacylate the O-tRNA with a natural amino acid. Thisprovides an O-RS that preferentially aminoacylates the O-tRNA with theunnatural amino acid. A nucleic acid that encodes the O-tRNA and anucleic acid that encodes the O-RS (or the components O-tRNA and/orO-RS) are introduced into a eukaryotic cell of a second species e.g., amammal, an insect, a fungus, an algae, a plant and/or the like.Typically, the O-tRNA is obtained by subjecting to negative selection apopulation of eukaryotic cells of a first species, where the eukaryoticcells comprise a member of a library of tRNAs. The negative selectioneliminates cells that comprise a member of the library of tRNAs that isaminoacylated by an aminoacyl-tRNA synthetase (RS) that is endogenous tothe eukaryotic cells, which provides a pool of tRNAs that are orthogonalto the eukaryotic cell of the first species and the second species.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofthe protein biosynthetic machinery. For example, a selector codonincludes, e.g., a unique three base codon, a nonsense codon, such as astop codon, e.g., an amber codon (UAG), an opal codon (UGA), anunnatural codon, at least a four base codon, a rare codon, or the like.A number of selector codons can be introduced into a desired gene, e.g.,one or more, two or more, more than three, etc. Once gene can includemultiple copies of a given selector codon, or can include multipledifferent selector codons, or any combination thereof.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivoin a eukaryotic cell. For example, an O-tRNA is produced that recognizesthe stop codon, e.g., UAG, and is aminoacylated by an O-RS with adesired unnatural amino acid. This O-tRNA is not recognized by thenaturally occurring host's aminoacyl-tRNA synthetases. Conventionalsite-directed mutagenesis can be used to introduce the stop codon, e.g.,TAG, at the site of interest in a polypeptide of interest. See, e.g.,Sayers, J. R., et al. (1988), 5′,3′ Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis. NucleicAcids Res, 791-802. When the O-RS, O-tRNA and the nucleic acid thatencodes the polypeptide of interest are combined in vivo, the unnaturalamino acid is incorporated in response to the UAG codon to give apolypeptide containing the unnatural amino acid at the specifiedposition.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, e.g., the amber suppressor tRNA, and aeukaryotic release factor (e.g., eRF) (which binds to a stop codon andinitiates release of the growing peptide from the ribosome), thesuppression efficiency can be modulated by, e.g., increasing theexpression level of O-tRNA, e.g., the suppressor tRNA.

Selector codons also comprise extended codons, e.g., four or more basecodons, such as, four, five, six or more base codons. Examples of fourbase codons include, e.g., AGGA, CUAG, UAGA, CCCU and the like. Examplesof five base codons include, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, e.g., one or multiple unnatural amino acids into the sameprotein. For example, in the presence of mutated O-tRNAs, e.g., aspecial frameshift suppressor tRNAs, with anticodon loops, e.g., with atleast 8-10 nt anticodon loops, the four or more base codon is read assingle amino acid. In other embodiments, the anticodon loops can decode,e.g., at least a four-base codon, at least a five-base codon, or atleast a six-base codon or more. Since there are 256 possible four-basecodons, multiple unnatural amino acids can be encoded in the same cellusing a four or more base codon. See, Anderson et al., (2002) Exploringthe Limits of Codon and Anticodon Size, Chemistry and Biology,9:237-244; Magliery, (2001) Expanding the Genetic Code: Selection ofEfficient Suppressors of Four-base Codons and Identification of “Shifty”Four-base Codons with a Library Approach in Escherichia coli, J. Mol.Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in invention, which canreduce missense readthrough and frameshift suppression at other unwantedsites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11586; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274. A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Unnatural Amino Acids

As used herein, an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand/or pyrrolysine and the following twenty genetically encodedalpha-amino acids: alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine. The generic structure of an alpha-aminoacid is illustrated by Formula I:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of theinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain, the unnatural amino acids formamide bonds with other amino acids, e.g., natural or unnatural, in thesame manner in which they are formed in naturally occurring proteins.However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amine, and the like, or any combination thereof. Otherunnatural amino acids of interest include, but are not limited to, aminoacids comprising a photoactivatable cross-linker, spin-labeled aminoacids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, biotin or biotin-analogue containing amino acids, keto containingamino acids, amino acids comprising polyethylene glycol or polyether,heavy atom substituted amino acids, chemically cleavable orphotocleavable amino acids, amino acids with an elongated side chain ascompared to natural amino acids (e.g., polyethers or long chainhydrocarbons, e.g., greater than about 5, greater than about 10 carbons,etc.), carbon-linked sugar-containing amino acids, redox-active aminoacids, amino thioacid containing amino acids, and amino acids containingone or more toxic moiety. In some embodiments, the unnatural amino acidshave a photoactivatable cross-linker that is used, e.g., to link aprotein to a solid support. In one embodiment, the unnatural amino acidshave a saccharide moiety attached to the amino acid side chain (e.g.,glycosylated amino acids) and/or other carbohydrate modification.

In addition to unnatural amino acids that contain novel side chains,unnatural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula H and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3,4,6,7,8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

For example, many unnatural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like.Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, where the substitutedtyrosine comprises, e.g., a keto group (e.g., an acetyl group), abenzoyl group, an amino group, a hydrazine, an hydroxyamine, a thiolgroup, a carboxy group, an isopropyl group, a methyl group, a C₆-C₂₀straight chain or branched hydrocarbon, a saturated or unsaturatedhydrocarbon, an O-methyl group, a polyether group, a nitro group, analkynyl group or the like. In addition, multiply substituted aryl ringsare also contemplated. Glutamine analogs of the invention include, butare not limited to, α-hydroxy derivatives, γ-substituted derivatives,cyclic derivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, e.g., a hydroxy group,a methoxy group, a methyl group, an allyl group, an aldehyde, an azido,an iodo, a bromo, a keto group (e.g., an acetyl group), a benzoyl, analkynyl group, or the like. Specific examples of unnatural amino acidsinclude, but are not limited to, a p-acetyl-L-phenylalanine, ap-propargyloxyphenylalanine, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, and the like. Examples of structures ofunnatural amino acids are illustrated in FIG. 7, Panel B and FIG. 11.Additional structures of a variety of unnatural amino acids are providedin, for example; FIGS. 16, 17, 18, 19, 26, and 29 of WO 2002/085923entitled “In vivo incorporation of unnatural amino acids.” See also,FIG. 1 structures 2-5 of Kiick et al., (2002) Incorporation of azidesinto recombinant proteins for chemoselective modification ^(,) by theStaudinger ligtation, PNAS 99:19-24, for additional methionine analogs.

In one embodiment, compositions that include an unnatural amino acid(such as p-(propargyloxy)-phenyalanine) are provided. Variouscompositions comprising p-(propargyloxy)-phenyalanine and, e.g.,proteins and/or cells, are also provided. In one aspect, a compositionthat includes the p-(propargyloxy)-phenyalanine unnatural amino acidfurther includes an orthogonal tRNA. The unnatural amino acid can bebonded (e.g., covalently) to the orthogonal tRNA, e.g., covalentlybonded to the orthogonal tRNA though an amino-acyl bond, covalentlybonded to a 3′OH or a 2′OH of a terminal ribose sugar of the orthogonaltRNA, etc.

The chemical moieties via an unnatural amino acids that can beincorporated into proteins offer a variety of advantages andmanipulations of the protein. For example, the unique reactivity of aketo functional group allows selective modification of proteins with anyof a number of hydrazine- or hydroxylamine-containing reagents in vitroand in vivo. A heavy atom unnatural amino acid, for example, can beuseful for phasing x-ray structure data. The site-specific introductionof heavy atoms using unnatural amino acids also provides selectivity andflexibility in choosing positions for heavy atoms. Photoreactiveunnatural amino acids (e.g., amino acids with benzophenone andarylazides (e.g., phenylazide) side chains), for example, allow forefficient in vivo and in vitro photocrosslinking of proteins. Examplesof photoreactive unnatural amino acids include, but are not limited to,e.g., p-azido-phenylalanine and p-benzoyl-phenylalanine. The proteinwith the photoreactive unnatural amino acids can then be crosslinked atwill by excitation of the photoreactive group-providing temporal (and/orspatial) control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, e.g., methyl group, asa probe of local structure and dynamics, e.g., with the use of nuclearmagnetic resonance and vibrational spectroscopy. Alkynyl or azidofunctional groups, for example, allow the selective modification ofproteins with molecules through a [3+2] cycloaddition reaction.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids provided above are commerciallyavailable, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA).Those that are not commercially available are optionally synthesized asprovided herein or as provided in various publications or using standardmethods known to those of skill in the art. For organic synthesistechniques, see, e.g., Organic Chemistry by Fessendon and Fessendon,(1982, Second Edition, Willard Grant Press, Boston Mass.); AdvancedOrganic Chemistry by March (Third Edition, 1985, Wiley and Sons, NewYork); and Advanced Organic Chemistry by Carey and Sundberg (ThirdEdition, Parts A and B, 1990, Plenum Press, New York). Additionalpublications describing the synthesis of unnatural amino acids include,e.g., WO 2002/085923 entitled “In vivo incorporation of Unnatural AminoAcids;” Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669; King, F.E. & Kidd, D. A. A. (1949) A New Synthesis of Glutamine and ofγ-Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem.Soc., 3315-3319; Friedman, O. M. & Chatterrji, R. (1959) Synthesis ofDerivatives of Glutamine as Model Substrates for Anti-Tumor Agents. J.Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al. (1988) AbsoluteConfiguration of the Enantiomers of7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials. Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 1989:1859-1866; Barton et al., (1987) Synthesis of Novela-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L-and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also, patentapplication Ser. No. 60/435,821 entitled “Protein Arrays,” filed on Dec.22, 2002.

In one aspect of the invention, a method for synthesizing ap-(propargyloxy)phenyalanine compound is provided. A method comprises,e.g., (a) suspending N-tert-butoxycarbonyl-tyrosine and K₂CO₃ inanhydrous DMF; (b) adding propargyl bromide to the reaction mixture of(a) and alkylating the hydroxyl and the carboxyl group, resulting in anprotected intermediate compound having the structure:

and (c) mixing the protected intermediate compound with anhydrous HCl inMeOH and deprotecting the amine moiety, thereby synthesizing thep-(propargyloxy)phenyalanine compound. In one embodiment, the methodfurther comprises (d) dissolving the p-(propargyloxy)phenylalanine HClin aqueous NaOH and MeOH and stirring it at room temperature; (e)adjusting the pH of to pH 7; and (f) precipitating thep-(propargyloxy)phenylalanine compound. See e.g., synthesis ofpropargyloxyphenylalanine in Example 4, herein.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a eukaryotic cell is one issue that istypically considered when designing and selecting unnatural amino acids,e.g., for incorporation into a protein. For example, the high chargedensity of α-amino acids suggests that these compounds are unlikely tobe cell permeable. Natural amino acids are taken up into the eukaryoticcell via a collection of protein-based transport systems. A rapid screencan be done which assesses which unnatural amino acids, if any, aretaken up by cells. See, e.g., the toxicity assays in, e.g., theapplication Ser. No. 60/435,821 entitled “Protein Arrays,” filed on Dec.22, 2002; and Liu, D. R. & Schultz, P. G. (1999) Progress toward theevolution of an organism with an expanded genetic code. PNAS UnitedStates 96:4780-4785. Although uptake is easily analyzed with variousassays, an alternative to designing unnatural amino acids that areamenable to cellular uptake pathways is to provide biosynthetic pathwaysto create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, e.g., in aeukaryotic cell, the invention provides such methods. For example,biosynthetic pathways for unnatural amino acids are optionally generatedin host cell by adding new enzymes or modifying existing host cellpathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, e.g., in Genbank. Artificially evolved enzymes arealso optionally added into a cell in the same manner. In this manner,the cellular machinery and resources of a cell are manipulated toproduce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, e.g., as developed by Maxygen, Inc.(available on the world wide web at www.maxygen.com), is optionally usedto develop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391;and, Stemmer, (1994), DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution, Proc. Natl.Acad. Sci. USA., 91:10747-10751. Similarly DesignPath™, developed byGenencor (available on the world wide web at genencor.com) is optionallyused for metabolic pathway engineering, e.g., to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,e.g., identified through functional genomics, and molecular evolutionand design. Diversa Corporation (available on the world wide web atdiversa.com) also provides technology for rapidly screening libraries ofgenes and gene pathways, e.g., to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, e.g., a natural cellularamount, but not to such a degree as to affect the concentration of theother amino acids or exhaust cellular resources. Typical concentrationsproduced in vivo in this manner are about 10 mM to about 0.05 mM. Once acell is transformed with a plasmid comprising the genes used to produceenzymes desired for a specific pathway and an unnatural amino acid isgenerated, in vivo selections are optionally used to further optimizethe production of the unnatural amino acid for both ribosomal proteinsynthesis and cell growth.

Polypeptides with Unnatural Amino Acids

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient (e.g.,a pharmaceutically acceptable excipient) can also be present with theprotein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic posttranslational modifications. In certainembodiments, a protein includes at least one unnatural amino acid and atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is not madeby a prokaryotic cell. For example, the post-translation modificationincludes, e.g., acetylation, acylation, lipid-modification,palmitoylation, palmitate addition, phosphorylation, glycolipid-linkagemodification, glycosylation, and the like. In one aspect, thepost-translational modification includes attachment of anoligosaccharide (e.g., (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to anasparagine by a GlcNAc-asparagine linkage. See also, Table 7, whichlists some examples of N-linked oligosaccharides of eukaryotic proteins(additional residues can also be present, which are not shown). Inanother aspect, the post-translational modification includes attachmentof an oligosaccharide (e.g., Gal-GalNAc, Gal-GlcNAc, etc.) to a serineor threonine by a GalNAc-serine or GalNAc-threonine linkage, or aGlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 7 EXAMPLES OF OLIGOSACCHARIDES THROUGH GlcNAc-LINKAGE Type BaseStructure High- mannose

Hybrid

Complex

Xylose

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (e.g., calcitonin precursor,calcitonin gene-related peptide precursor, preproparathyroid hormone,preproinsulin, proinsulin, prepro-opiomelanocortin, pro-opiomelanocortinand the like), assembly into a multisubunit protein or macromolecularassembly, translation to another site in the cell (e.g., to organelles,such as the endoplasmic reticulum, the golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic cell, or in vitro.Thus, in certain embodiments, the post-translational modification isthrough the unnatural amino acid. For example, the post-translationalmodification can be through a nucleophilic-electrophilic reaction. Mostreactions currently used for the selective modification of proteinsinvolve covalent bond formation between nucleophilic and electrophilicreaction partners, e.g. the reaction of α-haloketones with histidine orcysteine side chains. Selectivity in these cases is determined by thenumber and accessibility of the nucleophilic residues in the protein. Inproteins of the invention, other more selective reactions can be used,such as the reaction of an unnatural keto-amino acid with hydrazides oraminooxy compounds, in vitro and in vivo. See, e.g., Cornish, et al.,(1996) Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, in press. This allows the selective labeling ofvirtually any protein with a host of reagents including fluorophores,crosslinking agents, saccharide derivatives and cytotoxic molecules. Seealso, patent application U.S. Ser. No. 10/686,944 entitled “Glycoproteinsynthesis” filed Oct. 15, 2003. Post-translational modifications, e.g.,through an azido amino acid, can also made through the Staudingerligation (e.g., with triarylphosphine reagents). See, e.g., Kiick etal., (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligtation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, e.g., containing an azide oralkynyl moiety (see, e.g., 2 and 1 of FIG. 11), into proteins inresponse to a selector codon. These amino acid side chains can then bemodified by, e.g., a Huisgen [3+2] cycloaddition reaction (see, e.g.,Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost,B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in 1,3-DipolarCycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p.1-176) with, e.g., alkynyl or azide derivatives, respectively. See,e.g., FIG. 16. Because this method involves a cycloaddition rather thana nucleophilic substitution, proteins can be modified with extremelyhigh selectivity. This reaction can be carried out at room temperaturein aqueous conditions with excellent regioselectivity (1,4>1,5) by theaddition of catalytic amounts of Cu(I) salts to the reaction mixture.See, e.g., Tornoe, et al., (2002) Org. Chem. 67:3057-3064; and,Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Anothermethod that can be used is the ligand exchange on a bisarsenic compoundwith a tetracysteine motif, see, e.g., Griffin, et al., (1998) Science281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azido oralkynyl derivative. See, e.g., Example 3 and 5, herein. Such moleculesinclude, but are not limited to, dyes, fluorophores, crosslinkingagents, saccharide derivatives, polymers (e.g., derivatives ofpolyethylene glycol), photocrosslinkers, cytotoxic compounds, affinitylabels, derivatives of biotin, resins, beads, a second protein orpolypeptide (or more), polynucleotide(s) (e.g., DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. See,e.g., FIG. 13A, and Example 3 and 5, herein. These molecules can beadded to an unnatural amino acid with an alkynyl group, e.g.,p-propargyloxyphenylalanine, or azido group, e.g.,p-azido-phenylalanine, respectively. For example, see FIG. 13B and FIG.17A

In another aspect, the invention provides compositions including suchmolecules and methods of producing these molecules, e.g., azido dyes(such as shown in chemical structure 4 and chemical structure 6), analkynyl polyethylene glycol (e.g., as shown in chemical structure 7),where n is an integer between, e.g., 50 and 10,000, 75 and 5,000, 100and 2,000, 100 and 1,000, etc. In embodiment of the invention, thealkynyl polyethylene glycol has a molecular weight of, e.g., about 5,000to about 100,000 Da, about 20,000 to about 50,000 Da, about 20,000 toabout 10,000 Da (e.g., 20,000 Da), etc.

Various compositions comprising these compounds, e.g., with proteins andcells, are also provided. In one aspect of the invention, a proteincomprising an azido dye (e.g., of chemical structure 4 or chemicalstructure 6), further includes at least one unnatural amino acid (e.g.,an alkynyl amino acid), where the azido dye is attached to the unnaturalamino acid through a [3+2] cycloaddition.

In one embodiment, a protein comprises the alkynyl polyethylene glycolof chemical structure 7. In another embodiment, the composition furtherincludes at least one unnatural amino acid (e.g., an azido amino acid),wherein the alkynyl polyethylene glycol is attached to an unnaturalamino acid through a [3+2] cycloaddition.

Methods for synthesizing azido dyes are also provided. For example, onesuch method comprises: (a) providing a dye compound comprising asulfonyl halide moiety; (b) warming the dye compound to room temperaturein the presence of 3-azidopropylamine and triethylamine and coupling anamine moiety of the 3-azidopropylamine to the halide position of the dyecompound, thereby synthesizing the azido dye. In one example embodiment,the dye compound comprises dansyl chloride, and the azido dye comprisesthe composition of chemical structure 4. In one aspect, the methodfurther comprises purifying the azido dye from the reaction mixture.See, e.g., Example 5, herein.

In another example, a method for synthesizing an azido dye comprises (a)providing an amine-containing dye compound; (b) combining theamine-containing dye compound with a carbodiimide and4-(3-azidopropylcarbamoyl)-butyric acid in a suitable solvent, andcoupling a carbonyl group of the acid to the amine moiety of the dyecompound, thereby synthesizing the azido dye. In one embodiment, thecarbodiimine comprises 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDCI). In one aspect, the amine-containing dye comprisesfluoresceinamine, and the suitable solvent comprises pyridine. Forexample, the amine-containing dye optionally comprises fluoresceinamineand the azido dye optionally comprises the composition of chemicalstructure 6. In one embodiment, the method further comprises (c)precipitating the azido dye; (d) washing the precipitate with HCl; (e)dissolving the washed precipitate in EtOAc; and (f) precipitating theazido dye in hexanes. See, e.g., Example 5, herein.

Methods for synthesizing a propargyl amide polyethylene glycol are alsoprovided. For example, the method comprises reacting propargylamine withpolyethylene glycol (PEG)-hydroxysuccinimide ester in an organic solvent(e.g., CH₂Cl₂) at room temperature, resulting in the propargyl amidepolyethylene glycol of chemical structure 7. In one embodiment, themethod further comprises precipitating the propargylamide polyethyleneglycol using ethyl acetate. In one aspect, the method further includesrecrystallizing the propargylamide polyethylene glycol in methanol; anddrying the product under a vacuum. See, e.g., Example 5, herein.

A eukaryotic cell of the invention provides the ability to synthesizeproteins that comprise unnatural amino acids in large useful quantities.In one aspect, the composition optionally includes, e.g., at least 10micrograms, at least 50 micrograms, at least 75 micrograms, at least 100micrograms, at least 200 micrograms, at least 250 micrograms, at least500 micrograms, at least 1 milligram, at least 10 milligrams or more ofthe protein that comprises an unnatural amino acid, or an amount thatcan be achieved with in vivo protein production methods (details onrecombinant protein production and purification are provided herein). Inanother aspect, the protein is optionally present in the composition ata concentration of, e.g., at least 10 micrograms of protein per liter,at least 50 micrograms of protein per liter, at least 75 micrograms ofprotein per liter, at least 100 micrograms of protein per liter, atleast 200 micrograms of protein per liter, at least 250 micrograms ofprotein per liter, at least 500 micrograms of protein per liter, atleast 1 milligram of protein per liter, or at least 10 milligrams ofprotein per liter or more, in, e.g., a cell lysate, a buffer, apharmaceutical buffer, or other liquid suspension (e.g., in a volume of,e.g., anywhere from about 1 n1 to about 100 L). The production of largequantities (e.g., greater that that typically possible with othermethods, e.g., in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

The incorporation of an unnatural amino acid can be done to, e.g.,tailor changes in protein structure and/or function, e.g., to changesize, acidity, nucleophilicity, hydrogen bonding, hydrophobicity,accessibility of protease target sites, target to a moiety (e.g., for aprotein array), etc. Proteins that include an unnatural amino acid canhave enhanced or even entirely new catalytic or physical properties. Forexample, the following properties are optionally modified by inclusionof an unnatural amino acid into a protein: toxicity, biodistribution,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic ability, half-life (e.g., serumhalf-life), ability to react with other molecules, e.g., covalently ornoncovalently, and the like. The compositions including proteins thatinclude at least one unnatural amino acid are useful for, e.g., noveltherapeutics, diagnostics, catalytic enzymes, industrial enzymes,binding proteins (e.g., antibodies), and e.g., the study of proteinstructure and function. See, e.g., Dougherty, (2000) Unnatural AminoAcids as Probes of Protein Structure and Function, Current Opinion inChemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, or at least ten or more unnatural amino acids. The unnaturalamino acids can be the same or different, e.g., there can be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different unnaturalamino acids. In another aspect, a composition includes a protein with atleast one, but fewer than all, of a particular amino acid present in theprotein is substituted with the unnatural amino acid. For a givenprotein with more than one unnatural amino acids, the unnatural aminoacids can be identical or different (e.g., the protein can include twoor more different types of unnatural amino acids, or can include two ofthe same unnatural amino acid). For a given protein with more than twounnatural amino acids, the unnatural amino acids can be the same,different or a combination of a multiple unnatural amino acid of thesame kind with at least one different unnatural amino acid.

Essentially any protein (or portion thereof) that includes an unnaturalamino acid (and any corresponding coding nucleic acid, e.g., whichincludes one or more selector codons) can be produced using thecompositions and methods herein. No attempt is made to identify thehundreds of thousands of known proteins, any of which can be modified toinclude one or more unnatural amino acid, e.g., by tailoring anyavailable mutation methods to include one or more appropriate selectorcodon in a relevant translation system. Common sequence repositories forknown proteins include GenBank EMBL, DDBJ and the NCBI. Otherrepositories can easily be identified by searching the internet.

Typically, the proteins are, e.g., at least 60%, at least 70%, at least75%, at least 80%, at least 90%, at least 95%, or at least 99% or moreidentical to any available protein (e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof, and thelike), and they comprise one or more unnatural amino acid. Examples oftherapeutic, diagnostic, and other proteins that can be modified tocomprise one or more unnatural amino acids include, but are not limitedto, e.g., Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor,antibodies (further details on antibodies are found below),Apolipoprotein, Apoprotein, Atrial natriuretic factor, Atrialnatriuretic polypeptide, Atrial peptides, C—X—C chemokines (e.g.,T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1,PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractantprotein-1, Monocyte chemoattractant protein-2, Monocyte chemoattractantprotein-3, Monocyte inflammatory protein-1 alpha, Monocyte inflammatoryprotein-1 beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065,T64262), CD40 ligand, C-kit Ligand, Collagen, Colony stimulating factor(CSF), Complement factor 5a, Complement inhibitor, Complement receptor1, cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-1δ, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”, representing a preferred target formodification by the incorporation of one or more unnatural amino acid),Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, FactorX, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF,GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehogproteins (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte GrowthFactor (HGF), Hirudin, Human serum albumin, Insulin, Insulin-like GrowthFactor (IGF), interferons (e.g., IFN-α, IFN-β, IFN-γ), interleukins(e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, etc.), Keratinocyte Growth Factor (KGF), Lactoferrin,leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitoryfactor (NIF), oncostatin M, Osteogenic protein, Parathyroid hormone,PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone),Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C,Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1,Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin,Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcalenterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Superoxidedismutase (SOD), Toxic shock syndrome toxin (TSST-1), Thymosin alpha 1,Tissue plasminogen activator, Tumor necrosis factor beta (TNF beta),Tumor necrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNFalpha), Vascular Endothelial Growth Factor (VEGEF), Urokinase, and manyothers.

One class of proteins that can be made using the compositions andmethods for in vivo incorporation of unnatural amino acids describedherein includes transcriptional modulators or portions thereof. Exampletranscriptional modulators include genes and transcriptional modulatorproteins that modulate cell growth, differentiation, regulation, or thelike. Transcriptional modulators are found in prokaryotes, viruses, andeukaryotes, including fungi, plants, yeasts, insects, and animals,including mammals, providing a wide range of therapeutic targets. Itwill be appreciated that expression and transcriptional activatorsregulate transcription by many mechanisms, e.g., by binding toreceptors, stimulating a signal transduction cascade, regulatingexpression of transcription factors, binding to promoters and enhancers,binding to proteins that bind to promoters and enhancers, unwinding DNA,splicing pre-mRNA, polyadenylating RNA, and degrading RNA. For example,compositions of GAL4 protein or portion thereof in a eukaryotic cell arealso a feature of the invention. Typically, the GAL4 protein or portionthereof comprises at least one unnatural amino acid. See also thesection herein entitled “Orthogonal aminoacyl-tRNA synthetases.”

One class of proteins of the invention (e.g., proteins with one or moreunnatural amino acids) include expression activators such as cytokines,inflammatory molecules, growth factors, their receptors, and oncogeneproducts, e.g., interleukins (e.g., IL-1, IL-2, IL-8, etc.),interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;signal transduction molecules and corresponding oncogene products, e.g.,Mos, Ras, Raf, and Met; and transcriptional activators and suppressors,e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptorssuch as those for estrogen, progesterone, testosterone, aldosterone, theLDL receptor ligand and corticosterone.

Enzymes (e.g., industrial enzymes), or portions thereof with at leastone unnatural amino acid, are also provided by the invention. Examplesof enzymes include, but are not limited to, e.g., amidases, amino acidracemases, acylases, dehalogenases, dioxygenases, diarylpropaneperoxidases, epimerases, epoxide hydrolases, esterases, isomerases,kinases, glucose isomerases, glycosidases, glycosyl transferases,haloperoxidases, monooxygenases (e.g., p450s), lipases, ligninperoxidases, nitrile hydratases, nitrilases, proteases, phosphatases,subtilisins, transaminase, and nucleases.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genbank). Any of them can be modified by theinsertion of one or more unnatural amino acid according to theinvention, e.g., to alter the protein with respect to one or moretherapeutic, diagnostic or enzymatic properties of interest. Examples oftherapeutically relevant properties include serum half-life, shelfhalf-life, stability, immunogenicity, therapeutic activity,detectability (e.g., by the inclusion of reporter groups (e.g., labelsor label binding sites) in the unnatural amino acids), reduction of LD₅₀or other side effects, ability to enter the body through the gastrictract (e.g., oral availability), or the like. Examples of diagnosticproperties include shelf half-life, stability, diagnostic activity,detectability, or the like. Examples of relevant enzymatic propertiesinclude shelf half-life, stability, enzymatic activity, productioncapability, or the like.

A variety of other proteins can also be modified to include one or moreunnatural amino acid of the invention. For example, the invention caninclude substituting one or more natural amino acids in one or morevaccine proteins with an unnatural amino acid, e.g., in proteins frominfectious fungi, e.g., Aspergillus, Candida species; bacteria,particularly E. coli, which serves a model for pathogenic bacteria, aswell as medically important bacteria such as Staphylococci (e.g.,aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as(+) RNA viruses (examples include Poxviruses e.g., vaccinia;Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses,e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g., Rhabdoviruses,e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza;Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, forexample), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV,and certain DNA to RNA viruses such as Hepatitis B.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targets forunnatural amino acid modification.

The invention also provides methods for producing in a eukaryotic cellat least one protein comprising at least one unnatural amino acid (andproteins produced by such methods). For example, a method includes:growing, in an appropriate medium, a eukaryotic cell that comprises anucleic acid that comprises at least one selector codon and encodes theprotein. The eukaryotic cell also comprises: an orthogonal tRNA (O-tRNA)that functions in the cell and recognizes the selector codon; and anorthogonal aminoacyl tRNA synthetase (O-RS) that preferentiallyaminoacylates the O-tRNA with the unnatural amino acid, and the mediumcomprises an unnatural amino acid.

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (e.g., a dye, a polymer, e.g., a derivative of polyethyleneglycol, a photocrosslinker, a cytotoxic compound, an affinity label, aderivative of biotin, a resin, a second protein or polypeptide, a metalchelator, a cofactor, a fatty acid, a carbohydrate, a polynucleotide(e.g., DNA, RNA, etc.), and the like) that comprises a second reactivegroup. The first reactive group reacts with the second reactive group toattach the molecule to the unnatural amino acid through a [3+2]cycloaddition. In one embodiment, the first reactive group is an alkynylor azido moiety and the second reactive group is an azido or alkynylmoiety. For example, the first reactive group is the alkynyl moiety(e.g., in unnatural amino acid p-propargyloxyphenylalanine) and thesecond reactive group is the azido moiety. In another example, the firstreactive group is the azido moiety (e.g., in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In one embodiment, the O-RS aminoacylates the O-tRNA with the unnaturalamino acid at least 50% as efficiently as does an O-RS having an aminoacid sequence, e.g., as set forth in SEQ ID NO.: 86 or 45. In anotherembodiment, the O-tRNA comprises, is processed from, or is encoded bySEQ ID NO.: 65 or 64, or a complementary polynucleotide sequencethereof. In yet another embodiment, the O-RS comprises an amino acid setforth in any one of SEQ ID NO.: 36-63 (e.g., 36-47, 48-63, or any othersubset of 36-63) and/or 86.

The encoded protein can comprise, e.g., a therapeutic protein, adiagnostic protein, an industrial enzyme, or portion thereof.Optionally, the protein that is produced by the method is furthermodified through the unnatural amino acid. For example, the proteinproduced by the method is optionally modified by at least onepost-translational modification in vivo.

Methods of producing a screening or selecting transcriptional modulatorprotein are also provided (and screening or selecting transcriptionalmodulator proteins produced by such methods). For example, a methodincludes: selecting a first polynucleotide sequence, where thepolynucleotide sequence encodes a nucleic acid binding domain; andmutating the first polynucleotide sequence to include at least oneselector codon. This provides a screening or selecting polynucleotidesequence. The method also includes: selecting a second polynucleotidesequence, where the second polynucleotide sequence encodes atranscriptional activation domain; providing a construct that comprisesthe screening or selecting polynucleotide sequence operably linked tothe second polynucleotide sequence; and, introducing the construct, anunnatural amino acid, an orthogonal tRNA synthetase (O-RS) and anorthogonal tRNA (O-tRNA) into a cell. With these components, the O-RSpreferentially aminoacylates the O-tRNA with the unnatural amino acidand the O-tRNA recognizes the selector codon and incorporates theunnatural amino acid into the nucleic acid binding domain, in responseto the selector codon in the screening or selecting polynucleotidesequence, thereby providing the screening or selecting transcriptionalmodulator protein.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinunder “Mutagenesis and Other Molecular Biology Techniques” to include,e.g., one or more selector codon for the incorporation of an unnaturalamino acid. For example, a nucleic acid for a protein of interest ismutagenized to include one or more selector codon, providing for theinsertion of the one or more unnatural amino acids. The inventionincludes any such variant, e.g., mutant, versions of any protein, e.g.,including at least one unnatural amino acid. Similarly, the inventionalso includes corresponding nucleic acids, i.e., any nucleic acid withone or more selector codon that encodes one or more unnatural aminoacid.

In one example embodiment, the invention provides compositions (&compositions produced by the methods of the invention) that include aThr44, Arg110 TAG mutant of GAL4, where the GAL4 protein includes atleast one unnatural amino acid. In another embodiment, the inventionprovides compositions that include a Trp33 TAG mutant of humanSuperoxide dimutase (hSOD), where the hSOD protein includes at least oneunnatural amino.

Purifying Recombinant Proteins Comprising Unnatural Amino Acids

Proteins of the invention, e.g., proteins comprising unnatural aminoacids, antibodies to proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods well known in the art, including,e.g., ammonium sulfate or ethanol precipitation, acid or baseextraction, column chromatography, affinity column chromatography, anionor cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxylapatite chromatography,lectin chromatography, gel electrophoresis and the like. Proteinrefolding steps can be used, as desired, in making correctly foldedmature proteins. High performance liquid chromatography (HPLC), affinitychromatography or other suitable methods can be employed in finalpurification steps where high purity is desired. In one embodiment,antibodies made against unnatural amino acids (or proteins comprisingunnatural amino acids) are used as purification reagents, e.g., foraffinity-based purification of proteins comprising one or more unnaturalamino acid(s). Once purified, partially or to homogeneity, as desired,the polypeptides are optionally used e.g., as assay components,therapeutic reagents or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, N J, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic cell is that typically the proteinsor polypeptides will be folded in their native conformations. However,in certain embodiments of the invention, those of skill in the art willrecognize that, after synthesis, expression and/or purification,proteins can possess a conformation different from the desiredconformations of the relevant polypeptides. In one aspect of theinvention, the expressed protein is optionally denatured and thenrenatured. This is accomplished, e.g., by adding a chaperonin to theprotein or polypeptide of interest, and/or by solubilizing the proteinsin a chaotropic agent such as guanidine HCl, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are well knownto those of skill in the art (see, the references above, and Debinski,et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan(1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski, et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The proteins can be refolded in a redox buffer containing, e.g.,oxidized glutathione and L-arginine. Refolding reagents can be flowed orotherwise moved into contact with the one or more polypeptide or otherexpression product, or vice-versa.

Antibodies

In one aspect, the invention provides antibodies to molecules of theinvention, e.g., synthetases, tRNA, and proteins comprising unnaturalamino acids. Antibodies to molecules of the invention are useful aspurification reagents, e.g., for purifying the molecules of theinvention. In addition, the antibodies can be used as indicator reagentsto indicate the presence of a synthetase, a tRNA, or protein comprisingan unnatural amino acid, e.g., to track the presence or location (e.g.,in vivo or in situ) of the molecule.

An antibody of the invention can be a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (V_(H)) refer to these light and heavy chains,respectively.

Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab′)₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab′)₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting theF(ab′)₂dimer into an Fab′ monomer. The Fab′ monomer is essentially anFab with part of the hinge region (see, Fundamental Immunology, 4^(th)addition, W. E. Paul, ed., Raven Press, N.Y. (1999), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that such Fab′ fragments, etc. may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also optionallyincludes antibody fragments either produced by the modification of wholeantibodies or synthesized de novo using recombinant DNA methodologies.Antibodies include single chain antibodies, including single chain Fv(sFv or scFv) antibodies in which a variable heavy and a variable lightchain are joined together (directly or through a peptide linker) to forma continuous polypeptide. Antibodies of the invention can be, e.g.,polyclonal, monoclonal, chimeric, humanized, single chain, Fabfragments, fragments produced by an Fab expression library, or the like.

In general, antibodies of the invention are valuable, both as generalreagents and as therapeutic reagents in a variety of molecularbiological or pharmaceutical processes. Methods of producing polyclonaland monoclonal antibodies are available, and can be applied to makingthe antibodies of the invention. A number of basic texts describestandard antibody production processes, including, e.g., Borrebaeck (ed)(1995) Antibody Engineering, 2^(nd) Edition Freeman and Company, NY(Borrebaeck); McCafferty et al. (1996) Antibody Engineering, A PracticalApproach IRL at Oxford Press, Oxford, England (McCafferty), and Paul(1995) Antibody Engineering Protocols Humana Press, Towata, N.J. (Paul);Paul (ed.), (1999) Fundamental Immunology, Fifth edition Raven Press,N.Y.; Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; and Kohler andMilstein (1975) Nature 256: 495-497.

A variety of recombinant techniques for antibody preparation which donot rely on, e.g., injection of an antigen into an animal have beendeveloped and can be used in the context of the present invention. Forexample, it is possible to generate and select libraries of recombinantantibodies in phage or similar vectors. See, e.g., Winter et al. (1994)Making Antibodies by Phage Display Technology Annu. Rev. Immunol.12:433-55 and the references cited therein for a review. See also,Griffiths and Duncan (1998) Strategies for selection of antibodies byphage display Curr Opin Biotechnol 9: 102-8; Hoogenboom et al. (1998)Antibody phage display technology and its applications Immunotechnology4: 1-20; Gram et al. (1992) in vitro selection and affinity maturationof antibodies from a naïve combinatorial immunoglobulin library PNAS89:3576-3580; Huse et al. (1989) Science 246: 1275-1281; and Ward, etal. (1989) Nature 341: 544-546.

In one embodiment, antibody libraries can include repertoires of V genes(e.g., harvested from populations of lymphocytes or assembled in vitro)which are cloned for display of associated heavy and light chainvariable domains on the surface of filamentous bacteriophage. Phage areselected by binding to an antigen. Soluble antibodies are expressed fromphage infected bacteria and the antibody can be improved, e.g., viamutagenesis. See e.g., Balint and Larrick (1993) Antibody Engineering byParsimonious Mutagenesis Gene 137:109-118; Stemmer et al. (1993)Selection of an Active Single Chain Fv Antibody From a Protein LinkerLibrary Prepared by Enzymatic Inverse PCR Biotechniques 14(2):256-65;Crameri et al. (1996) Construction and evolution of antibody-phagelibraries by DNA shuffling Nature Medicine 2:100-103; and Crameri andStemmer (1995) Combinatorial multiple cassette mutagenesis creates allthe permutations of mutant and wildtype cassettes BioTechniques18:194-195.

Kits for cloning and expression of recombinant antibody phage systemsare also known and available, e.g., the “recombinant phage antibodysystem, mouse ScFv module,” from Amersham-Pharmacia Biotechnology(Uppsala, Sweden). Bacteriophage antibody libraries have also beenproduced for making high affinity human antibodies by chain shuffling(See, e.g., Marks et al. (1992) By-Passing Immunization: Building HighAffinity Human Antibodies by Chain Shuffling Biotechniques 10:779-782.It will also be recognized that antibodies can be prepared by any of anumber of commercial services (e.g., Bethyl Laboratories (Montgomery,Tex.), Anawa (Switzerland), Eurogentec (Belgium and in the US inPhiladelphia, Pa., etc.) and many others.

In certain embodiments, it is useful to “humanize” antibodies of theinvention, e.g., where the antibodies are to be administeredtherapeutically. The use of humanized antibodies tends to reduce theincidence of unwanted immune responses against the therapeuticantibodies (e.g., when the patient is a human). The antibody referencesabove describe humanization strategies. In addition to humanizedantibodies, human antibodies are also a feature of the invention. Humanantibodies consist of characteristically human immunoglobulin sequences.Human antibodies can be produced in using a wide variety of methods(see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, for a review). Ageneral approach for producing human antibodies by trioma technology isdescribed by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.

A variety of methods of using antibodies in the purification anddetection of proteins are known and can be applied to detecting andpurifying proteins comprising unnatural amino acids as noted herein. Ingeneral, antibodies are useful reagents for ELISA, western blotting,immunochemistry, affinity chromatograpy methods, SPR, and many othermethods. The references noted above provide details on how to performELISA assays, western blots, surface plasmon resonance (SPR) and thelike.

In one aspect of the invention, antibodies of the invention themselvesinclude unnatural amino acids, providing the antibodies with propertiesof interest (e.g., improved half-life, stability, toxicity, or thelike). See also, the section herein entitled “Polypeptides withunnatural amino acids.” Antibodies account for nearly 50% of allcompounds currently in clinical trials (Wittrup, (1999) Phage on displayTibtech 17: 423-424 and antibodies are used ubiquitously as diagnosticreagents. Accordingly, the ability to modify antibodies with unnaturalamino acids provides an important tool for modifying these valuablereagents.

For example, there are many applications of MAbs to the field ofdiagnostics. Assays range from simple spot tests to more involvedmethods such as the radio-labeled NR-LU-10 MAb from DuPont Merck Co.used for tumor imaging (Rusch et al. (1993) NR-LU-10 monoclonal antibodyscanning. A helpful new adjunct to computed tomography in evaluatingnon-small-cell lung cancer. J Thorac Cardiovasc Surg 106: 200-4). Asnoted, MAbs are central reagents for ELISA, western blotting,immunochemistry, affinity chromatograpy methods and the like. Any suchdiagnostic antibody can be modified to include one or more unnaturalamino acid, altering, e.g., the specificity or avidity of the Ab for atarget, or altering one or more detectable property, e.g., by includinga detectable label (e.g., spectrographic, fluorescent, luminescent,etc.) in the unnatural amino acid.

One class of valuable antibody reagents are therapeutic Abs. Forexample, antibodies can be tumor-specific MAbs that arrest tumor growthby targeting tumor cells for destruction by antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-mediated lysis (CML)(these general types of Abs are sometimes referred to as “magicbullets”). One example is Rituxan, an anti-CD20 MAb for the treatment ofNon-Hodgkins lymphoma (Scott (1998) Rituximab: a new therapeuticmonoclonal antibody for non-Hodgkin's lymphoma Cancer Pract 6: 195-7). Asecond example relates to antibodies which interfere with a criticalcomponent of tumor growth. Herceptin is an anti-HER-2 monoclonalantibody for treatment of metastatic breast cancer, and provides anexample of an antibody with this mechanism of action (Baselga et al.(1998) Recombinant humanized anti-HER2 antibody (Herceptin) enhances theantitumor activity of paclitaxel and doxorubicin against HER2/neuoverexpressing human breast cancer xenografts [published erratum appearsin Cancer Res (1999) 59(8):2020], Cancer Res 58: 2825-31). A thirdexample relates to antibodies for delivery of cytotoxic compounds(toxins, radionuclides, etc.) directly to a tumor or other site ofinterest. For example, one application Mab is CYT-356, a 90Y-linkedantibody that targets radiation directly to prostate tumor cells (Deb etal. (1996) Treatment of hormone-refractory prostate cancer with90Y-CYT-356 monoclonal antibody Clin Cancer Res 2: 1289-97. A fourthapplication is antibody-directed enzyme prodrug therapy, where an enzymeco-localized to a tumor activates a systemically-administered pro-drugin the tumor vicinity. For example, an anti-Ep-CAM1 antibody linked tocarboxypeptidase A is being developed for treatment of colorectal cancer(Wolfe et al. (1999) Antibody-directed enzyme prodrug therapy with theT268G mutant of human carboxypeptidase A1: in vitro and in vivo studieswith prodrugs of methotrexate and the thymidylate synthase inhibitorsGW1031 and GW1843 Bioconjug Chem 10: 38-48). Other Abs (e.g.,antagonists) are designed to specifically inhibit normal cellularfunctions for therapeutic benefit. An example is Orthoclone OKT3, ananti-CD3 MAb offered by Johnson and Johnson for reducing acute organtransplant rejection (Strate et al. (1990) Orthoclone OKT3 as first-linetherapy in acute renal allograft rejection Transplant Proc 22: 219-20.Another class of antibody products are agonists. These Mabs are designedto specifically enhance normal cellular functions for therapeuticbenefit. For example, Mab-based agonists of acetylcholine receptors forneurotherapy are under development (Xie et al. (1997) Directdemonstration of MuSK involvement in acetylcholine receptor clusteringthrough identification of agonist ScFv Nat. Biotechnol. 15: 768-71. Anyof these antibodies can be modified to include one or more unnaturalamino acid to enhance one or more therapeutic property (specificity,avidity, serum-half-life, etc.).

Another class of antibody products provide novel functions. The mainantibodies in this group are catalytic antibodies such as Ig sequencesthat have been engineered to mimic the catalytic abilities of enzymes(Wentworth and Janda (1998) Catalytic antibodies Curr Opin Chem Biol 2:138-44. For example, an interesting application involves using thecatalytic antibody mAb-15A10 to hydrolyze cocaine in vivo for addictiontherapy (Mets et al. (1998) A catalytic antibody against cocaineprevents cocaine's reinforcing and toxic effects in rats Proc Natl AcadSci USA 95: 10176-81). Catalytic antibodies can also be modified toinclude one or more unnatural amino acid to improve one or more propertyof interest.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising unnatural amino acids in thecase of proteins synthesized in the translation systems herein, or,e.g., in the case of the novel synthetases herein, novel sequences ofstandard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antibodies or antibodies which specifically bind thepolypeptides of the invention, as well as the polypeptides which arebound by such antibodies or antisera, are a feature of the invention.

For example, the invention includes synthetase proteins thatspecifically bind to or that are specifically immunoreactive with anantibody or antisera generated against an immunogen comprising an aminoacid sequence selected from one or more of (SEQ ID NO: 36-63 (e.g.,36-47, 48-63, or any other subset of 36-63), and/or 86). To eliminatecross-reactivity with other homologues, the antibody or antisera issubtracted with available control synthetase homologues, such as thewild-type E. coli tyrosyl synthetase (TyrRS) (e.g., SEQ ID NO.:2).

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to one or more of SEQ ID NO: 36-63 (e.g., 36-47,48-63, or any other subset of 36-63), and/or 86, or a substantialsubsequence thereof (i.e., at least about 30% of the full lengthsequence provided). The set of potential polypeptide immunogens derivedfrom SEQ ID NO: 36-63 and 86 are collectively referred to below as “theimmunogenic polypeptides.” The resulting antisera is optionally selectedto have low cross-reactivity against the control synthetase homologuesand any such cross-reactivity is removed, e.g., by immunoabsorbtion,with one or more control synthetase homologues, prior to use of thepolyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional references and discussion of antibodies is also found hereinand can be applied here to make antibodies that define/detectpolypeptides by immunoreactivity). Alternatively, one or more syntheticor recombinant polypeptide derived from the sequences disclosed hereinis conjugated to a carrier protein and used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control synthetase polypeptidesto produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologues in a comparative immunoassay.In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic synthetase ascompared to binding to a control synthetase homologue. That is, thestringency of the binding/washing reaction(s) is/are adjusted by theaddition of non-specific competitors such as albumin or non-fat drymilk, and/or by adjusting salt conditions, temperature, and/or the like.These binding/washing conditions are used in subsequent assays fordetermining whether a test polypeptide (a polypeptide being compared tothe immunogenic polypeptides and/or the control polypeptides) isspecifically bound by the pooled subtracted polyclonal antisera. Inparticular, test polypeptides which show at least a 2-5× higher signalto noise ratio than the control synthetase homologue underdiscriminatory binding conditions, and at least about a ½ signal tonoise ratio as compared to the immunogenic polypeptide(s), sharessubstantial structural similarity with the immunogenic polypeptide ascompared to known synthetases, and is, therefore a polypeptide of theinvention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is optionally determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptides and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Pharmacetutical Compositions

The polypeptides or proteins of the invention (e.g., synthetases,proteins comprising one or more unnatural amino acid, etc.) areoptionally employed for therapeutic uses, e.g., in combination with asuitable pharmaceutical carrier. Such compositions, e.g., comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier or excipient includes,but is not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and/or combinations thereof. The formulation is madeto suit the mode of administration. In general, methods of administeringproteins are well known in the art and can be applied to administrationof the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can be initially determined by activity,stability or other suitable measures of unnatural herein to naturalamino acid homologues (e.g., comparison of an EPO modified to includeone or more unnatural amino acids to a natural amino acid EPO), i.e., ina relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Unnatural amino acid polypeptide compositions can also beadministered via liposomes. Such administration routes and appropriateformulations are generally known to those of skill in the art.

The unnatural amino acid polypeptide, alone or in combination with othersuitable components, can also be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (e.g.,those typically used for EPO, GCSF, GMCSF, IFNs, interleukins,antibodies, and/or any other pharmaceutically delivered protein), alongwith formulations in current use, provide preferred routes ofadministration and formulation for the proteins that include unnaturalamino acids of the invention (e.g., pegylated variants of currentthereputic proteins, etc.).

The dose administered to a patient, in the context of the presentinvention, is sufficient to effect a beneficial therapeutic response inthe patient over time, or, e.g., to inhibit infection by a pathogen, orother appropriate activity, depending on the application. The dose isdetermined by the efficacy of a particular composition/formulation, andthe activity, stability or serum half-life of the unnatural amino acidpolypeptide employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularcomposition/formulation, or the like in a particular patient.

In determining the effective amount of the composition/formulation to beadministered in the treatment or prophylaxis of disease (e.g., cancers,inherited diseases, diabetes, AIDS, or the like), the physicianevaluates circulating plasma levels, formulation toxicities, progressionof the disease, and/or where relevant, the production of anti-unnaturalamino acid polypeptide antibodies.

The dose administered, e.g., to a 70 kilogram patient, is typically inthe range equivalent to dosages of currently-used therapeutic proteins,adjusted for the altered activity or serum half-life of the relevantcomposition. The compositions/formulations of this invention cansupplement treatment conditions by any known conventional therapy,including antibody administration, vaccine administration,administration of cytotoxic agents, natural amino acid polypeptides,nucleic acids, nucleotide analogues, biologic response modifiers, andthe like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acids at various concentrations, e.g., as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistamines. Treatment is slowedor discontinued depending upon the severity of the reaction.

Nucleic Acid and Polypeptide Sequence and Variants

As described above and below, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,O-tRNAs and O-RSs, and, e.g., compositions and methods comprising saidsequences. Examples of said sequences, e.g., O-tRNAs and O-RSs aredisclosed herein (see, Table 5, e.g., SEQ ID NO. 3-65, 86, and otherthan SEQ ID NO.: 1 and 2). However, one of skill in the art willappreciate that the invention is not limited to those sequencesdisclosed herein, e.g., the Examples and Table 5. One of skill willappreciate that the invention also provides many related and evenunrelated sequences with the functions described herein, e.g., encodingan O-tRNA or an O-RS.

The invention also provides polypeptides (O-RSs) and polynucleotides,e.g., O-tRNA, polynucleotides that encode O-RSs or portions thereof(e.g., the active site of the synthetase), oligonucleotides used toconstruct aminoacyl-tRNA synthetase mutants, etc. For example, apolypeptide of the invention includes a polypeptide that comprises anamino acid sequence as shown in any one of SEQ ID NO.: 36-63 (e.g.,36-47, 48-63, or any other subset of 36-63), and/or 86, a polypeptidethat comprises an amino acid sequence encoded by a polynucleotidesequence as shown in any one of SEQ ID NO.: 3-35 (e.g., 3-19, 20-35, orany other subset of sequences 3-35), and a polypeptide that isspecifically immunoreactive with an antibody specific for a polypeptidethat comprises an amino acid sequence as shown in any one of SEQ ID NO.:36-63, and/or 86, or a polypeptide that comprises an amino acid sequenceencoded by a polynucleotide sequence as shown in any one of SEQ ID NO.:3-35 (e.g., 3-19, 20-35, or any other subset of sequences 3-35).

Also included among the polypeptides of the invention are polypeptidesthat comprise an amino acid sequence that is at least 90% identical tothat of a naturally occurring tyrosyl aminoacyl-tRNA synthetase (TyrRS)(e.g., SEQ ID NO.:2) and comprises two or more amino acids of groupsA-E. For example, group A includes valine, isoleucine, leucine, glycine,serine, alanine, or threonine at a position corresponding to Tyr37 of E.coli TyrRS; group B includes aspartate at a position corresponding toAsn126 of E. coli TyrRS; group C includes threonine, serine, arginine,asparagine or glycine at a position corresponding to Asp182 of E. coliTyrRS; group D includes methionine, alanine, valine, or tyrosine at aposition corresponding to Phe183 of E. coli TyrRS; and, group E includesserine, methionine, valine, cysteine, threonine, or alanine at aposition corresponding to Leu186 of E. coli TyrRS. Any subset ofcombinations of these groups are a feature of the invention. Forexample, in one embodiment, the O-RS has two or more amino acidsselected from valine, isoleucine, leucine, or threonine occurs at aposition corresponding to Tyr37 of E. coli TyrRS; threonine, serine,arginine, or glycine at a position corresponding to Asp182 of E. coliTyrRS; methionine, or tyrosine at a position corresponding to Phe183 ofE. coli TyrRS; and, serine, or alanine at a position corresponding toLeu186 of E. coli TyrRS. In another embodiment, the O-RS includes twomore amino acids selected from glycine, serine, or alanine at a positioncorresponding to Tyr37 of E. coli TyrRS, aspartate at a positioncorresponding to Asn126 of E. coli TyrRS, asparagine at a positioncorresponding to Asp182 of E. coli TyrRS, alanine, or valine, at aposition corresponding to Phe183 of E. coli TyrRS, and/or methionine,valine, cysteine, or threonine, at a position corresponding to Leu186 ofE. coli TyrRS. Similarly, polypeptides of the invention also include apolypeptide that comprises at least 20 contiguous amino acids of SEQ IDNO.: 36-63 (e.g., 36-47, 48-63, or any other subset of 36-63), and/or86, and two or more amino acid substitutions as indicated above ingroups A-E. See also, Table 4, Table 6, and/or Table 8, herein. An aminoacid sequence comprising a conservative variation of any of the abovepolypeptides is also included as a polypeptide of the invention.

In one embodiment, a composition includes a polypeptide of the inventionand an excipient (e.g., buffer, water, pharmaceutically acceptableexcipient, etc.). The invention also provides an antibody or antiseraspecifically immunoreactive with a polypeptide of the invention.

Polynucleotides are also provided in the invention. Polynucleotides ofthe invention include those that encode proteins or polypeptides ofinterest of the invention, or that include one or more selector codon,or both. For example, polynucleotides of the invention include, e.g., apolynucleotide comprising a nucleotide sequence as set forth in any oneof SEQ ID NO.: 3-35 (e.g., 3-19, 20-35, or any other subset of sequences3-35), 64-85; a polynucleotide that is complementary to or that encodesa polynucleotide sequence thereof; and/or a polynucleotide encoding apolypeptide that comprises an amino acid sequence as set forth in anyone of SEQ ID NO.: 36-63, and/or 86, or a conservative variationthereof. A polynucleotide of the invention also includes apolynucleotide that encodes a polypeptide of the invention. Similarly, anucleic acid that hybridizes to a polynucleotide indicated above underhighly stringent conditions over substantially the entire length of thenucleic acid is a polynucleotide of the invention.

A polynucleotide of the invention also includes a polynucleotide thatencodes a polypeptide that comprises an amino acid sequence that is atleast 90% identical to that of a naturally occurring tyrosylaminoacyl-tRNA synthetase (TyrRS) (e.g., SEQ ID NO.: 2) and comprisestwo or more mutations as indicated above in groups A-E (above). Apolynucleotide that is that is at least 70%, (or at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or least99% or more) identical to a polynucleotide indicated above and/or apolynucleotide comprising a conservative variation of any of thepolynucleotides indicated above are also included among thepolynucleotides of the invention. See also, Table 4, Table 6, and/orTable 8, herein.

In certain embodiments, a vector (e.g., a plasmid, a cosmid, a phage, avirus, etc.) comprises a polynucleotide of the invention. In oneembodiment, the vector is an expression vector. In another embodiment,the expression vector includes a promoter operably linked to one or moreof the polynucleotides of the invention. In another embodiment, a cellcomprises a vector that includes a polynucleotide of the invention.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid. Thus, “conservative variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup. Finally, the addition of sequences that do not alter the encodedactivity of a nucleic acid molecule, such as the addition of anon-functional sequence, is a conservative variation of the basicnucleic acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following sets forth example groupswhich contain natural amino acids that include “conservativesubstitutions” for one another.

Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T)2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NO: 3-35 (e.g., 3-19, 20-35, or any other subset of sequences3-35), 64-85 under high, ultra-high and ultra-ultra high stringencyconditions are a feature of the invention. Examples of such nucleicacids include those with one or a few silent or conservative nucleicacid substitutions as compared to a given nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at lest ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York), aswell as in Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRLPress at Oxford University Press, Oxford, England, (Hames and Higgins 1)and Hames and Higgins (1995) Gene Probes 2 IRL Press at OxfordUniversity Press, Oxford, England (Hames and Higgins 2) provide detailson the synthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria are met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In one aspect, the invention provides a nucleic acid that comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNAs and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the invention were derived,e.g., by mutation). Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, preferably 80%, mostpreferably 90-95% nucleotide or amino acid residue identity, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Mutagenesis and Other Molecular Biology Techniques

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thegeneration of genes that include selector codons for production ofproteins that include unnatural amino acids, orthogonal tRNAs,orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the invention, e.g., to producelibraries of tRNAs, to produce libraries of synthetases, to insertselector codons that encode unnatural amino acids in a protein orpolypeptide of interest. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination, DNAshuffling or other recursive mutagenesis methods, chimeric construction,mutagenesis using uracil containing templates, oligonucleotide-directedmutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesisusing gapped duplex DNA or the like, or any combination thereof.Additional suitable methods include point mismatch repair, mutagenesisusing repair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,e.g., involving chimeric constructs, are also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like.

The above texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462(1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201(1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.154: 329-350 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787(1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698(1986); Sayers et al., Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802(1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984); Carter et al., Improved oligonucleotide site-directedmutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985);Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors,Methods in Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Useof oligonucleotides to generate large deletions, Nucl. Acids Res. 14:5115 (1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundstromet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001). W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

The invention also relates to eukaryotic host cells and organisms forthe in vivo incorporation of an unnatural amino acid via orthogonaltRNA/RS pairs. Host cells are genetically engineered (e.g., transformed,transduced or transfected) with the polynucleotides of the invention orconstructs which include a polynucleotide of the invention, e.g., avector of the invention, which can be, for example, a cloning vector oran expression vector. The vector can be, for example, in the form of aplasmid, a bacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (From etal., Proc. Natl. Acad. Sci. USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, e.g. for cell isolation and culture (e.g., for subsequentnucleic acid isolation) include Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, FL.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria, (see, e.g., EasyPrep™, FlexiPrep™, both from PharmaciaBiotech; StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). Theisolated and purified plasmids are then further manipulated to produceother plasmids, used to transfect cells or incorporated into relatedvectors to infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and integration in prokaryotes, eukaryotes,or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, etal., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr. Purif.6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue ofBacteria and Bacteriophages useful for cloning is provided, e.g., by theATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992)Gherna et al. (eds) published by the ATCC. Additional basic proceduresfor sequencing, cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY. Inaddition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asthe Midland Certified Reagent Company (Midland, Tex. mcrc.com), TheGreat American Gene Company (Ramona, Calif. available on the World WideWeb at genco.com), ExpressGen Inc. (Chicago, Ill. available on the WorldWide Web at expressgen.com), Operon Technologies Inc. (Alameda, Calif.)and many others.

Kits

Kits are also a feature of the invention. For example, a kit forproducing a protein that comprises at least one unnatural amino acid ina cell is provided, where the kit includes a container containing apolynucleotide sequence encoding an O-tRNA, and/or an O-tRNA, and/or apolynucleotide sequence encoding an O-RS, and/or an O-RS. In oneembodiment, the kit further includes at least one unnatural amino acid.In another embodiment, the kit further comprises instructional materialsfor producing the protein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. One of skill will recognize a variety of non-criticalparameters that may be altered without departing from the scope of theclaimed invention.

Example 1 Methods of Producing and Compositions of Aminoacyl-tRNASynthetases that Incorporate Unnatural Amino Acids in Eukaryotic Cells

The expansion of the eukaryotic genetic code to include unnatural aminoacids with novel physical, chemical or biological properties wouldprovide powerful tools for analyzing and controlling protein function inthese cells. Towards this goal, a general approach for the isolation ofaminoacyl-tRNA synthetases that incorporate unnatural amino acids withhigh fidelity into proteins in response to an amber codon inSaccharomyces cerevisiae (S. cerevisiae) is described. The method isbased on the activation of GAL4 responsive reporter genes, HIS3, URA3 orLacZ, by suppression of amber codons between the DNA binding domain andtranscriptional activation domain of GAL4. The optimization of a GAL4reporter for positive selection of active Escherichia coli tyrosyl-tRNAsynthetase (EcTyrRS) variants is described. A negative selection ofinactive EcTyrRS variants has also been developed with the URA3 reporterby use of a small molecule (5-fluroorotic acid (5-FOA)) added to thegrowth media as a ‘toxic allele.’ Importantly both positive and negativeselections can be performed on a single cell and with a range ofstringencies. This can facilitate the isolation of a range ofaminoacyl-tRNA synthetase (aaRS) activities from large libraries ofmutant synthetases. The power of the method for isolating desired aaRSphenotypes is demonstrated by model selections.

The recent addition of unnatural amino acids to the genetic code ofEscherichia coli (E. coli) provides a powerful new approach foranalyzing and manipulating protein structure and function both in vitroand in vivo. Amino acids with photoaffinity labels, heavy atoms, ketoand olefinic groups and chromophores have been incorporated intoproteins in E. coli with an efficiency and fidelity rivaling that of thecommon twenty amino acids. See, e.g., Chin, et al., (2002), Addition ofa Photocrosslinker to the Genetic Code of Escherichia coli, Proc. Natl.Acad. Sci. U.S.A. 99:11020-11024; Chin and Schultz, (2002), In vivoPhotocrosslinking with Unnatural Amino Acid Mutagenesis, Chem BioChem11:1135-1137; Chin et al., (2002), Addition of p-Azido-L-phenylalanineto the Genetic code of Escherichia coli, J. Am. Chem. Soc.124:9026-9027; Zhang et al., (2002), The selective incorporation ofalkenes into proteins in Escherichia coli, Angewandte Chemie.International Ed. in English 41:2840-2842; and, Wang and Schultz,(2002), Expanding the Genetic Code, Chem. Comm. 1-10.

Unnatural amino acids have been introduced previously into the nicotinicacetylcholine receptor in Xenopus oocytes (e.g., M. W. Nowak, et al.(1998), In vivo incorporation of unnatural amino acids into ion channelsin Xenopus oocyte expression system, Method Enzymol. 293:504-529) bymicroinjection of a chemically misacylated Tetrahymena thermophila tRNA(e.g., M. E. Saks, et al. (1996), An engineered Tetrahymena tRNAGln forin vivo incorporation of unnatural amino acids into proteins by nonsensesuppression, J. Biol. Chem. 271:23169-23175, and the relevant mRNA. Thishas allowed detailed biophysical studies of the receptor in oocytes bythe introduction of amino acids containing side chains with uniquephysical or chemical properties. See, e.g., D. A. Dougherty (2000),Unnatural amino acids as probes of protein structure and function, Curr.Opin. Chem. Biol. 4:645-652. Unfortunately, this methodology is limitedto proteins in cells that can be microinjected, and because the tRNA ischemically acylated in vitro, and cannot be re-acylated, the yields ofprotein are very low. This in turn necessitates sensitive techniques toassay protein function.

There is interest in the genetic incorporation of unnatural amino acidsinto proteins in eukaryotic cells in response to an amber codon. Seealso, H. J. Drabkin et al., (1996), Amber suppression in mammalian cellsdependent upon expression of an Escherichia coli aminoacyl-tRNAsynthetase gene, Molecular & Cellular Biology 16:907-913; A. K. Kowal,et al., (2001), Twenty-first aminoacyl-tRNA synthetase-suppressor tRNApairs for possible use in site-specific incorporation of amino acidanalogues into proteins in eukaryotes and in eubacteria.[comment], Proc.Natl. Acad. Sci. U.S.A. 98:2268-2273; and, K. Sakamoto, et al., (2002),Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells, Nucleic Acids Res. 30:4692-4699. This would havesignificant technical and practical advantages, since tRNAs would bere-acylated by their cognate synthetases-leading to large amounts ofmutant protein. Moreover, genetically encoded aminoacyl-tRNA synthetasesand tRNAs are, in principle, heritable, allowing the unnatural aminoacid to be incorporated into proteins through many cell divisionswithout exponential dilution.

The steps necessary to add new amino acids to the genetic code of E.coli have been described (see, e.g., D. R. Liu, & P. G. Schultz, (1999),Progress toward the evolution of an organism with an expanded geneticcode, Proc. Natl. Acad. Sci. U.S.A. 96:4780-4785; and similar principlescan be useful for expanding the genetic code of eukaryotes. In the firststep, an orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA_(CUA) pair isidentified. This pair needs to function with the host cellstranslational machinery, but the aaRS should not charge any endogenoustRNAs with an amino acid and the tRNA_(CUA) should not be aminoacylatedby any endogenous synthetases. See, e.g., D. R. Liu, et al., Engineeringa tRNA and aminoacyl-tRNA synthetase for the site-specific incorporationof unnatural amino acids into proteins in vivo, Proc. Natl. Acad. Sci.U.S.A. 94:10092-10097. In a second step, those aaRS/tRNA pairs that arecapable of using only the unnatural amino acid are selected from alibrary of mutant aaRSs. In E. coli the selection of unnatural aminoacid utilizing variants of MjTyrRS was carried out using two-step‘double sieve’ selections. See, e.g., D. R. Liu, & P. G. Schultz,(1999), Progress toward the evolution of an organism with an expandedgenetic code, Proc. Natl. Acad. Sci. U.S.A. 96:4780-4785. A modifiedselection method is used in eukaryotic cells.

Saccharomyces cerevisiae (S. cerevisiae) was chosen as the eukaryotichost organism, as it is unicellular, has a rapid generation time, aswell as relatively well characterized genetics. See, e.g., D. Burke, etal., (2000) Methods in Yeast Genetics. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY. Moreover, since the translationalmachinery of eukaryotes is highly conserved (see, e.g., (1996)Translational Control. Cold Spring Harbor Laboratory, Cold SpringHarbor, NY; Y. Kwok, & J. T. Wong, (1980), Evolutionary relationshipbetween Halobacterium cutirubrum and eukaryotes determined by use ofaminoacyl-tRNA synthetases as phylogenetic probes, Canadian Journal ofBiochemistry 58:213-218; and, (2001) The Ribosome. Cold Spring harborLaboratory Press, Cold Spring Harbor, N.Y.), it is likely that aaRSsgenes for the incorporation of unnatural amino acids discovered in S.cerevisiae can be ‘cut and pasted’ into higher eukaryotic organisms andused, in partnership with cognate tRNAs (see, e.g., K. Sakamoto, et al.,(2002) Site-specific incorporation of an unnatural amino acid intoproteins in mammalian cells, Nucleic Acids Res. 30:4692-4699; and, C.Kohrer, et al., (2001), Import of amber and ochre suppressor tRNAs intomammalian cells: a general approach to site-specific insertion of aminoacid analogues into proteins, Proc. Natl. Acad. Sci. U.S.A.98:14310-14315) to incorporate unnatural amino acids. The expansion ofthe genetic code of S. cerevisiae is therefore a gateway to expandingthe genetic code of complex multicellular eukaryotic organisms. See,e.g., M. Buvoli, et al., (2000), Suppression of nonsense mutations incell culture and mice by multimerized suppressor tRNA genes, Molecular &Cellular Biology 20:3116-3124. The tyrosyl pair derived fromMethanococcus jannaschii TyrRS (MjTyrRS)/tRNA (see e.g., L. Wang, & P.G. Schultz, (2002), Expanding the Genetic Code, Chem. Comm. 1-10) whichwas previously used to expand the genetic code of E. coli is notorthogonal in eukaryotic organisms (e.g., P. Fechter, et al., (2001),Major tyrosine identity determinants in Methanococcus jannaschii andSaccharomyces cerevisiae tRNA(Tyr) are conserved but expresseddifferently, Eur. J. Biochem. 268:761-767) and a new orthogonal pair isrequired to expand the eukaryotic genetic code. Schimmel and coworkershave shown that the E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA_(CUA)pair suppresses amber codons in S. cerevisiae, and that E. colitRNA_(CUA) is not charged by endogenous aminoacyl tRNA synthetases inthe yeast cytosol (FIG. 2). See also, e.g., H. Edwards, et al., (1991),An Escherichia coli tyrosine transfer RNA is a leucine-specific transferRNA in the yeast Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. U.S.A.88:1153-1156; and, H. Edwards, & P. Schimmel (1990), A bacterial ambersuppressor in Saccharomyces cerevisiae is selectively recognized by abacterial aminoacyl-tRNA synthetase, Molecular & Cellular Biology10:1633-1641. In addition, EcTyrRS has been shown not to charge yeasttRNA in vitro. See, e.g., Y. Kwok, & J. T. Wong, (1980), Evolutionaryrelationship between Halobacterium cutirubrum and eukaryotes determinedby use of aminoacyl-tRNA synthetases as phylogenetic probes, CanadianJournal of Biochemistry 58:213-218; B. P. Doctor, et al., (1966),Studies on the species specificity of yeast and E. coli tyrosine tRNAs,Cold Spring Harbor Symp. Quant. Biol. 31:543-548; and, K. Wakasugi, etal., (1998), Genetic code in evolution: switching species-specificaminoacylation with a peptide transplant, EMBO Journal 17:297-305. Thus,the EcTyrRS/tRNA_(CUA) pair is a candidate for an orthogonal pair in S.cerevisiae, as well as in higher eukaryotes (e.g., A. K. Kowal, et al.,(2001), Twenty-first aminoacyl-tRNA synthetase-suppressor tRNA pairs forpossible use in site-specific incorporation of amino acid analogues intoproteins in eukaryotes and in eubacteria.[comment], Proc. Natl. Acad.Sci. U.S.A. 98 (2001) 2268-2273).

To broaden the substrate specificity of EcTyrRS in E. coli, Nishimuraand coworkers screened an error prone PCR generated library of mutantsof EcTyrRS and discovered a mutant with an improved ability toincorporate 3-azatyrosine. See, e.g., F. Hamano-Takaku, et al., (2000),A mutant Escherichia coli tyrosyltRNA synthetase utilizes the unnaturalamino acid azatyrosine more efficiently than tyrosine, J. Biol. Chem.275:40324-40328. However, this amino acid is incorporated throughout theproteome of E. coli, and the evolved enzyme still prefers tyrosine as asubstrate. Yokoyama and coworkers screened a small collection ofdesigned active site variants of EcTyrRS in a wheat germ translationsystem and discovered an EcTyrRS variant that utilizes 3-iodotyrosinemore effectively than tyrosine. See, D. Kiga, et al., (2002), Anengineered Escherichia coli tyrosyl-tRNA synthetase for site-specificincorporation of an unnatural amino acid into proteins in eukaryotictranslation and its application in a wheat germ cell free system, Proc.Natl. Acad. Sci. U.S.A. 99:9715-9720. In contrast to the enzymes we haveevolved in E. coli (e.g., J. W. Chin, et al., (2002), Addition of aPhotocrosslinker to the Genetic Code of Escherichia coli, Proc. Natl.Acad. Sci. U.S.A. 99:11020-11024; J. W. Chin, et al., (2002), Additionof p-Azido-L-phenylalanine to the Genetic code of Escherichia coli, J.Am. Chem. Soc. 124:9026-9027; L. Wang, et al., (2001), Expanding thegenetic code of Escherichia coli, Science 292:498-500; and, L. Wang, etal., (2002), Adding L-3-(2-naphthyl)alanine to the genetic code ofE-coli, J. Am. Chem. Soc. 124:1836-1837), this enzyme still incorporatestyrosine in the absence of the unnatural amino acid. See, e.g., D. Kiga,et al., (2002), An engineered Escherichia coli tyrosyl-tRNA synthetasefor site-specific incorporation of an unnatural amino acid into proteinsin eukaryotic translation and its application in a wheat germ cell freesystem, Proc. Natl. Acad. Sci. U.S.A. 99:9715-9720. Recently, Yokoyamaand coworkers have also demonstrated that this EcTyrRS mutant functionswith a tRNA_(CUA) from Bacillus stearothermophilus to suppress ambercodons in mammalian cells. See, K. Sakamoto, et al., (2002),Site-specific incorporation of an unnatural amino acid into proteins inmammalian cells, Nucleic Acids Res. 30:4692-4699.

A requirement is that any amino acid added to the eukaryotic geneticcode be incorporated with a fidelity similar to that of the commontwenty amino acids. To accomplish this goal, a general, in vivoselection method has been used for the discovery of EcTyrRSRNA_(CUA)variants that function in S. cerevisiae to incorporate unnatural aminoacids, but none of the common amino acids, in response to the ambercodon TAG. A major advantage of a selection is that enzymes whichselectively incorporate unnatural amino acids can be rapidly selectedand enriched from libraries of 10⁸ EcTyrRS active site variants, 6-7orders of magnitude more diversity than has been screened in vitro. See,e.g., D. Kiga, et al., (2002), An engineered Escherichia colityrosyl-tRNA synthetase for site-specific incorporation of an unnaturalamino acid into proteins in eukaryotic translation and its applicationin a wheat germ cell free system, Proc. Natl. Acad. Sci. U.S.A.99:9715-9720. This increase in diversity vastly increases the likelyhoodof isolating EcTyrRS variants for the incorporation of a diverse rangeof useful functionality with very high fidelity. See, e.g., L. Wang, &P. G. Schultz, (2002), Expanding the Genetic Code, Chem. Comm. 1-10.

To extend the selection approach to S. cerevisiae, the transcriptionalactivator protein, GAL4 was used (see FIG. 1). See, e.g., A. Laughon, etal., (1984), Identification of two proteins encoded by the Saccharomycescerevisiae GAL4 gene, Molecular & Cellular Biology 4:268-275; A.Laughon, & R. F. Gesteland, (1984), Primary structure of theSaccharomyces cerevisiae GAL4 gene, Molecular & Cellular Biology4:260-267; L. Keegan, et al., (1986), Separation of DNA binding from thetranscription-activating function of a eukaryotic regulatory protein,Science 231:699-704; and, M. Ptashne, (1988), How eukaryotictranscriptional activators work, Nature 335:683-689. The N-terminal 147amino acids of this 881 amino acid protein form a DNA binding domain(DBD) that binds DNA sequence specifically. See, e.g., M. Carey, et al.,(1989), An amino-terminal fragment of GAL4 binds DNA as a dimer, J. Mol.Biol. 209:423-432; and, E. Giniger, et al., (1985), Specific DNA bindingof GAL4, a positive regulatory protein of yeast, Cell 40:767-774. TheDBD is linked, by an intervening protein sequence, to a C-terminal 113amino acid activation domain (AD) that can activate transcription whenbound to DNA. See, e.g., J. Ma, & M. Ptashne, (1987), Deletion analysisof GAL4 defines two transcriptional activating segments, Cell48:847-853: and, J. Ma, & M. Ptashne, (1987), The carboxy-terminal 30amino acids of GAL4 are recognized by GAL80, Cell 50:137-142. Weenvisioned that by placing amber codons towards the N-terminal DBD of asingle polypeptide that contained both the N-terminal DBD of GAL4 andits C-terminal AD, amber suppression by the EcTyrRS/tRNA_(CUA) pair canbe linked to transcriptional activation by GALA (FIG. 1, Panel A). Bythe choice of appropriate GALA activated reporter genes both positiveand negative selections can be performed with the gene (FIG. 1, PanelB). While many reporter genes based on complementing the amino acidauxotrophy of a cell can be used for positive selections (eg: URA3,LEU2, HIS3, LYS2), the HIS3 gene is an attractive reporter gene, as theactivity of the protein it encodes (imidazole glycerol phosphatedehydratase) can be modulated in a dose dependent manner by addition of3-aminotriazole (3-AT). See, e.g., G. M. Kishore, & D. M. Shah, (1988),Amino acid biosynthesis inhibitors as herbicides, Annual Review ofBiochemistry 57:627-663. In S. cerevisiae, fewer genes have been usedfor negative selections. One of several negative selection strategies(see, e.g., A. J. DeMaggio, et al., (2000), The yeast split-hybridsystem, Method Enzymol. 328:128-137; H. M. Shih, et al., (1996), Apositive genetic selection for disrupting protein-protein interactions:identification of CREB mutations that prevent association with thecoactivator CBP, Proc. Natl. Acad. Sci. U.S.A. 93:13896-13901; M. Vidal,et al., (1996), Genetic characterization of a mammalian protein-proteininteraction domain by using a yeast reverse two-hybrid system.[comment],Proc. Natl. Acad. Sci. U. S. A. 93:10321-10326; and, M. Vidal, et al.,(1996), Reverse two-hybrid and one-hybrid systems to detect dissociationof protein-protein and DNA-protein interactions.[commend], Proc. Natl.Acad. Sci. U.S.A. 93:10315-10320) that has been successfully used is theURA3/5-fluroorotic acid (5-FOA) negative selection (e.g., J. D. Boeke,et al., (1984), A positive selection for mutants lackingorotidine-5′-phosphate decarboxylase activity in yeast: 5-fluorooroticacid resistance, Molecular & General Genetics 197:345-346) systemdescribed in the ‘reverse two-hybrid’ system developed by Vidal andco-workers. See, M. Vidal, et al., (1996), Genetic characterization of amammalian protein-protein interaction domain by using a yeast reversetwo-hybrid system.[comment], Proc. Natl. Acad. Sci. U. S. A.93:10321-10326; and, M. Vidal, et al., (1996), Reverse two-hybrid andone-hybrid systems to detect dissociation of protein-protein andDNA-protein interactions.[comment], Proc. Natl. Acad. Sci. U.S.A.93:10315-10320). In the reverse two-hybrid system, a genomicallyintegrated URA3 reporter is placed under a tightly controlled promoterthat contains GAL4 DNA binding sites. When two proteins that interactare produced as fusions to the GALA DBD and GAL4 AD they reconstitutethe activity of GALA and activate transcription of URA3. In the presenceof 5-FOA, the URA3 gene product converts 5-FOA to a toxic product,killing the cell. See, J. D. Boeke, et al., supra. This selection hasbeen used to select for proteins that disrupt a protein-proteininteraction and for mutations that disrupt a protein-proteininteraction. A variant for screening small molecule inhibitors ofprotein-protein interactions has also been described. See, e.g., J.Huang, & S. L. Schreiber, (1997) A yeast genetic system for selectingsmall molecule inhibitors of protein-protein interactions innanodroplets, Proc. Natl. Acad. Sci. U.S.A. 94:13396-13401.

The appropriate choice of amber codons in full length GAL4 allowsefficient positive selections for active EcTyrRS variants using either aHIS3 or URA3 GAL4 activated reporters to complement histidine or uracilauxotrophy in yeast cells. Moreover, the URA3 reporter can be used innegative selections for inactive EcTyrRS variants in the presence of5-FOA. In addition, colorometric assays using lacZ can be used to readout aminoacyl-tRNA synthetase activity in yeast cells.

Results and Discussion

The EcTyrRS gene was expressed under the control of the constitutiveADH1 promoter, and the tRNA_(CUA) gene was expressed from the same highcopy yeast plasmid (pEcTyrRStRNA_(CUA), FIG. 1, Panel C). Uponco-transformation of pEcTyrRStRNA_(CUA) and a low copy reporter thatcontains a single amber mutation between the DNA binding domain andactivation domain of a chimericGAL4 construct into MaV203, cells grew onselective media lacking histidine and containing 10-20 mM 3-AT (FIG. 2).When MaV203 cells were transformed with the same GAL4 construct andeither an inactive synthetase mutant (A5) or a construct lacking theEctRNA gene, no growth was observed on 10 mM 3-AT (FIG. 2). Theseexperiments establish that EcTyrRS can be constitutively expressed in afunctional form from the ADH1 promoter, that there is minimal endogenousamber suppression in MaV203, and that there is little charging ofEctRNA_(CUA) by yeast synthetases in this system. See, e.g., H. Edwards,et al., (1991), An Escherichia coli tyrosine transfer RNA is aleucine-specific transfer RNA in the yeast Saccharomyces cerevisiae,Proc. Natl. Acad. Sci. U.S.A. 88:1153-1156; and, H. Edwards, & P.Schimmel, (1990), A bacterial amber suppressor in Saccharomycescerevisiae is selectively recognized by a bacterial aminoacyl-tRNAsynthetase, Molecular & Cellular Biology 10:1633-1641. Since EcTyrRSdoes not charge S. cerevisiae tRNA (e.g., Y. Kwok, & J. T. Wong, (1980),Evolutionary relationship between Halobacterium cutirubrum andeukaryotes determined by use of aminoacyl-tRNA synthetases asphylogenetic probes, Canadian Journal of Biochemistry 58:213-218; B. P.Doctor, et al., (1966), Studies on the species specificity of yeast andE. coli tyrosine tRNAs, Cold Spring Harbor Symp. Quant. Biol.31:543-548; and, K. Wakasugi, et al., (1998), Genetic code in evolution:switching species-specific aminoacylation with a peptide transplant,EMBO Journal 17:297-305), these experiments confirm thatEcTyrRS/EctRNA_(CUA) are an orthogonal pair in S. cerevisiae.

While the first generation GALA chimera was able to activatetranscription of the weak HIS3 reporter it was unable to activatetranscription of the URA3 reporter in MaV203 sufficiently to allowsignificant growth on concentrations of 3-AT greater than 20 mM, or on-URA plates (FIG. 2). For the purposes of selection of EcTyrRS, variantsa second generation GALA construct was made. This GALA reporter wasdesigned to be more active, to have a greater dynamic range, and toavoid the accumulation of revertants. To increase the activity of theGAL4 reporters, full length GAL4 was used (which has a transcriptionalactivation activity twice that of a DBD-AD fusion (see, e.g., J. Ma, &M. Ptashne, (1987), Deletion analysis of GAL4 defines twotranscriptional activating segments, Cell 48:847-853) under the controlof a strong ADH1 promoter, and a high copy 2-μm plasmid (with a copynumber 10-30 times that of the centromeric plasmid of the initial GAL4chimera) was used. An increase in both the copy number of the plasmidand the activity of the protein it encodes should extend the dynamicrange of the reporters. Amber mutations were targeted to the region ofthe GALA gene that encodes amino acid residues 2 and 147 (FIG. 3). Thisregion is sufficient for sequence specific DNA binding (see, e.g., M.Carey, et al., (1989), An amino-terminal fragment of GAL4 binds DNA as adimer, J. Mol. Biol. 209:423-432), and lies to the 5′ side of the firstcryptic activation domain in the GAL4gene (see, e.g., J. Ma, & M.Ptashne, (1987) Deletion analysis of GAL4 defines two transcriptionalactivating segments, Cell 48:847-853), such that the truncated productsproduced in the absence of amber suppression are not anticipated toactivate transcription. The choice of amino acid codons to mutate wasguided by previous saturation mutagenesis selections on GALA (see, e.g.,M. Johnston, & J. Dover, (1988), Mutational analysis of the GAL4-encodedtranscriptional activator protein of Saccharomyces cerevisiae, Genetics120:63-74), as well as the X-ray structures of the N-terminal DNAbinding domain of GALA (see, e.g., R. Marmorstein, et al., (1992), DNArecognition by GAL4: structure of a protein-DNA complex.[comment],Nature 356:408-414; and, J. D. Baleja, et al., (1992), Solutionstructure of the DNA-binding domain of Cd2-GAL4 from S.cerevisiae[comment], Nature 356:450-453) and the NMR structure of itsdimerization region. See, e.g., P. Hidalgo, et al., (2001), Recruitmentof the transcriptional machinery through GAL11P: structure andinteractions of the GAL4 dimerization domain, Genes & Development15:1007-1020.

Full length GAL4 was cloned into a small pUC based vector to allow therapid construction of 10 single amber mutants (at the codons for aminoacids L3, I13, T44, F68, R110, V114, T121, I127, S131, T145) by sitedirected mutageneisis. GAL 4 and the resulting amber mutants were thensubcloned into a 2-μm yeast vector under the control of the full lengthADH1 promoter to create pGADGAL4 and a series of amber mutants denotedpGADGAL4 (xxTAG) (FIG. 1, Panel C), where xx denotes the amino acidcodon in the GAL4 gene that was mutated to the amber codon. Each GAL4mutant was co-transformed with either EcTyrRS/tRNA_(CUA) orA5/tRNA_(CUA) into MaV203 cells, converting transformants to leucine andtryptophan protrophy. pGADGAL4 itself transformed with very lowefficiency (<10-3 times that of the GAL4 amber mutants) and ispresumably deleterious to MaV203 cells at such high copy; no such effectwas observed with the amber mutants of GAL4.

The phenotypes of GAL4 reporters, in the presence of an active or deadsynthetase, were assayed on -URA plates, and 0.1% 5-FOA plates (FIG. 3,Panel A). Five GAL4 mutants (L3TAG, I13TAG, T44TAG, F68TAG, S131TAG)grew on -URA plates and failed to grow on 0.1% 5-FOA in the presence ofeither a wild type or inactive EcTyrRS. In these amber mutants,endogenous suppression is apparently sufficient to push theEcTyrRS/tRNA_(CUA) mediated suppression beyond the dynamic range of theURA3 reporter in MaV203. Five GAL4 single amber mutants (R110TAG,V114TAG, T121TAG, I127TAG, T145TAG) grew in the absence of uracil and inthe presence EcTyrRS/tRNA_(CUA), (but not A5/tRNA_(CUA)) and showed thereverse phenotype on 5-FOA. These mutants show EcTyrRS dependentphenotypes that fall within the dynamic range of the URA3 reporter inMaV203. The cleanest EcTyrRS dependent phenotype on both -URA and 0.1%5-FOA was observed with the R110 TAG mutant of GAL4. However, thismutant showed some blue color in X-GAL assays when cotransformed withA5. To further improve the dynamic range, a series of six double ambermutants of GAL4 were made containing R110 TAG (FIG. 3, Panel B), (L3TAG,R110TAG; I13TAG, R110TAG; T44TAG, R110TAG; R110TAG, T121TAG; R110TAG,I127TAG; R110TAG, T145TAG). Four of these double mutants (I13TAG,R110TAG; R110TAG, T121TAG; R110TAG, I127TAG and T145TAG, R110TAG) wereunable to grow in the absence of uracil and grew on 0.1% 5-FOA. Thesedouble mutants have activities outside (below) the dynamic range of theplate assays. Two of the double mutants (L3TAG, R110TAG and T44TAG,R110TAG) grew in the presence of wild type EcTyrRS/tRNA_(CUA), but notwith A5/tRNA_(CUA) on -URA plates; these mutants also showed theexpected reciprocal phenotypes on 5-FOA. pGADGAL4 (T44TAG, R110TAG), themore active of these two GAL4 mutants, was selected for a more detailedcharacterization (FIG. 4). MaV203 containing pGADGAL4(T44TAG,R110TAG)/pEcTyrRS-tRNA_(CUA) were blue on X-GAL but the correspondingstrain containing pA5/tRNA_(CUA) was not. Similarly MaV203 containingpGADGAL4(T44TAG, R110TAG)/pEcTyrRS/tRNA_(CUA) grew robustly on plateswith 3-AT concentrations up to 75 mM, and on -URA plates but thecorresponding strain containing pA5/tRNA_(CUA) failed to grow on 10 mM3AT or in the absence of uracil. Taken together, the EcTyrRS dependentphenotypes of pGADGAL4(T44TAG, R110TAG) can span the dynamic range ofthe URA3, HIS3 and lacZ reporters in MaV203.

It was of interest to determine the activity of GAL4 mutants in whichT44 or R110 were substituted with amino acids other than tyrosine, sincethe ability to substitute varied amino acids without altering theactivity of GAL4 is likely to be useful for selection of mutantaminoacyl-tRNA synthetases that can incorporate unnatural amino acidsinto proteins. See, e.g., M. Pasternak, et al., (2000), A new orthogonalsuppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organismwith an expanded genetic code, Helvetica Chemica Acta 83:2277. A seriesof five mutants of residue T44 in GAL4, (T44Y, T44W, T44F, T44D, T44K)were constructed in pGADGAL4 (R110TAG), since pGADGAL4 is itself toxic.A similar series of mutants at position R110 in GAL4, (R110Y, R110W,R110F, R110D, R110K) in pGADGAL4(T44TAG) was constructed. These mutantsare biased towards the large hydrophobic amino acid side chains that weare interested in incorporating into proteins, but also contain apositively and negatively charged residue as a stringent test ofpermissiveness. Each mutant was co-transformed with pEcTyrRS/tRNA_(CUA)into MaV203 cells and leu+ trp+ isolates assayed for lacZ production byortho-nitrophenyl-β-D-galactopyranoside (ONPG) hydrolysis (FIG. 5). Thevariation in activity between cells containing GAL4 with different aminoacids substituted for either T44 or R110 was less than 3 fold in allcases. This minimal variability demonstrates the permissiveness of thesesites to amino acid substitution without altering the transcriptionalactivity of GAL4. As expected from the activity of the single ambermutants assayed on selective plates, mutants of T44 made in the GAL4(R110TAG) background lead to slower hydrolysis of ONPG than mutants ofR110 made in the GAL4(T44TAG) background.

Model enrichment studies were performed to examine the ability of thesystem to select an active synthetase from a large excess of inactivesynthetases (Table 1, Table 2, FIG. 6). This selection models theability to select active synthetases from a library of variants in thepresence of an unnatural amino acid. MaV203 cells containing theGAL4(T44, R110) and EcTyrRS/tRNA_(CUA) were mixed with a 10 to 10⁶ foldexcess of GAL4(T44TAG, R110TAG) and A5/tRNA_(CUA) as judged by bothOD₆₆₀, and the fraction of colonies that turned blue when plated onnonselective-leu, -trp media and assayed by X-GAL overlay. Those cellsable to survive on 50 mM 3-AT or in the absence of uracil were selected.The ratio of cells surviving on 3-AT or -URA that were blue in the X-GALassay to those that were white, when compared to the same ratio in theabsence of selection, clearly demonstrates that the positive selectionscan enrich active synthetases from dead synthetases by a factor >10⁵(Table 1). Measurement of accurate enrichments for starting ratiosgreater than 1:10⁵ was generally not possible, because no more than 10⁶cells can be conveniently plated without significant crosstalk betweencells leading to unreliable phenotypes.

TABLE 1 MODEL POSITIVE SELECTIONS FOR FUNCTIONAL EcTyrRS. StartingRatio,EcYRS:A5^(a) 1:10 1:10² 1:10³ 1:10⁴ 1:10⁵ Cell Dilution   10³   10³ 10²10³ 10 10³ 1 10³ -Leu, Trp (#blue^(b)) 1360 (81)  1262 (0)   >10³ (1)   1774 (0)   >10⁴ (—)  1092 (0)   >10⁴ (—)   1472 (0)   -Ura (#blue^(b))152 (152) 9 (9) 8 (8) 0 (—) 5 (5) 0 (—) 16 (14) 0 (—) -His + 50 mM3AT(#blue^(b)) 135 (135) 7 (7)  0 (—) 0 (—) 3 (3) 0 (—) 10 (10) 0 (—)Enrichment factor >10 >10² >10³ >10⁴ >10⁵ ^(a)Determined by OD₆₆₀ ^(b)OnX-GAL

TABLE 2 MODEL NEGATIVE SELECTIONS FOR NON-FUNCTIONAL EcTyrRS (A5).StartingRatio, A5:EcYRS^(a) 1:10 1:10² 1:10³ 1:10⁴ Cell Dilution   10³  10²    10² 10²  10 -Leu, Trp 353 (22) 1401 (31) 1336 (2) 1375 (0) >10⁴(#white^(b)) 0.1% 5-FOA  16 (16)  41 (41)   4 (4)   0 (—) 2 (2)(#white^(b)) Enrichment factor >10 >45 >600 >0.67 × 10⁴ ^(a)Determinedby OD₆₆₀ ^(b)On X-GAL

After a positive selection in the presence of unnatural amino acid, theselected cells will contain synthetases able to use natural amino acidsand those able to use an added unnatural amino acid. To isolate thosesynthetases capable of using only the unnatural amino acid, cellsencoding synthetases that use natural amino acids must be deleted fromthe selected clones. This can be accomplished with a negative selectionin which the unnatural amino acid is withheld and those synthetases thatfunction with a natural amino acid are removed. A model negativeselection was performed in an analogous manner to the model positiveselection. EcTyrRS/tRNA_(CUA) was mixed with a 10 to 10⁵ fold excess ofA5/tRNA_(CUA) and selection was performed on 0.1% 5-FOA. Comparison ofthe ratio of cells surviving on 0.1% 5-FOA that were white in the X-GALassay to those that were blue, to the same ratio under non-selectiveconditions (see Table 2) makes it clear that the negative selections canenrich dead synthetases from active synthetases by a factor of at least0.6×10⁴. Measurement of accurate enrichments for starting ratios greaterthan 1:10⁴ was generally not possible, because no more than 10⁵ cellscould be conveniently plated without significant crosstalk between cellsleading to unreliable phenotypes.

A general approach was developed that allows both positive selection ofaaRSs that recognize unnatural amino acids and negative selection ofaaRSs that recognize natural amino acids. By varying the stringencies ofthe selection, a variety of synthetase activities can be isolated.Application of this method to a model selection using variants ofEcTyrRS showed enrichments of greater than 10⁵ in a single round ofeither positive selection and greater than 0.6×10⁴ in a single round ofnegative selection. These observations suggest that this method canprovide rapid access to orthogonal aminoacyl-tRNA synthetases thatfunction to site-specifically incorporation unnatural amino acids with adiversity of side chains into proteins in S. cerevisiae. Moreover,enzymes evolved in S. cerevisiae can be used in higher eukaryotes.

Materials and Methods

Vector Construction

The tRNA_(CUA) gene was amplified by PCR using the primerstRNA5′:GGGGGGACCGGTGGGGGGACCGGTAAGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGCATTACCCCGTGGTGGGTTCCCGA (SEQ ID NO:89), andtRNA3′:GGCGGCGCTAGCAAGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGGGAAGTTCAGGGACTTTTGAAAAAAATGGTGGTGGGGGAAGGAT (SEQ ID NO:90) frompESCSU3URA. This, and all other PCR reactions were preformed using theExpand PCR kit from Roche, according to the manufacturers instructions.After restriction endonuclease digestion with NheI and AgeI this tRNAgene was inserted between the same sites in the 2 μm vector pESCTrp(Stratagene) to yield ptRNA_(CUA). The full length ADH1 promoter wasamplified by PCR from pDBLeu (Invitrogen) with the primers PADHf:IGGGGGGACCGGTIGGGGGGACCGGTCGGGATCGAAGAAATGATGGTAAATGA AATAGGAAATCAAGG(SEQ ID NO:91) and pADHR:GGGGGGGAATTCAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGCAGAA AAAGAAAC (SEQ IDNO:92), digested with AgeI and EcoRI. EcTyrRS was amplified with theprimers pESCTrp1:TCATAACGAGAATTCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCTCATAACGAGAATTCATGGCAAGCAGTAACTTG (SEQ ID NO:93) andpESCTrp2:TTACTACGTGCGGCCGCATGGCAAGCAGTAACTTGTTACTACGTGCGGCCGCTTATTTCCAGCAAATCAGAC (SEQ ID NO:94). The EcTyrRS PCR product weredigested with EcoRI and Not I. ptRNA_(CUA) was then digested with Age Iand Not I. A triple ligation of these three DNAs yieldedpEcTyrRS-tRNA_(CUA). Plasmid pA5-tRNA_(CUA) in which amino acid residues(37,126,182,183 and 186 in the active site are mutated to alanine) wascreated by overlap PCR using the oligonucleotides F37Afwd:CCGATCGCGCTCGCTTGCGGCTTCGATC (SEQ ID NO:95), N126Afwd:ATCGCGGCGAACGCCTATGACTGGTTC (SEQ ID NO:96), 182,183,186A,GTTGCAGGGTTATGCCGCCGCCTGTGCGAACAAACAGTAC (SEQ ID NO:97) and theirreverse complements, as well as the flanking oligonucleotides, 4783:GCCGCTTTGCTATCAAGTATAAATAG (SEQ ID NO:98), 3256: CAAGCCGACAACCTTGATTGG(SEQ ID NO:99) and pEcTyrRS-tRNA_(CUA) as a template. The PCR productwas digested with EcoRI and Not I and ligated into the large fragment ofpEcTyrRS-tRNA_(CUA) released upon digestion with the same enzymes. Toconstruct 1st generation DB-AD reporters, the GAL4 DNA binding domainwas PCR amplified from pGADT7 (Clontech) using the forward primerpADfwd: GGGGACAAGTTTGTACAAAAAAGCAGGCTACGCCAATTTTAATCAAAGTGG GAATATTGC(SEQ ID NO:100) or pADfwd(TAG)GGGGACAAGTTTGTACAAAAAAGCAGGCTAGGCCAATTTTAATCAAAGTGG GAATATTGC (SEQ IDNO:101) and ADrev: GGGGACCACTTTGTACAAGAAAGCTGGGTTACTCTTTTTTTGGGTTTGGTGGGGTATC (SEQ ID NO:102). These PCR products were cloned into the vectorpDEST3-2 (invitrogen) using the Clonase procedure, according to themanufacturer's instructions, yielding pDB-AD and pDB-(TAG)-AD. Toconstruct PGADGAL4 and variants, the GAL4 gene was amplified from pCL1(Clontech) by PCR using the primers ADH1428-1429 AAGCTATACCAAGCATACAATC(SEQ ID NO:103), and GAL4C:ACAAGGCCTTGCTAGCTTACTCTTTTTTTGGGTTTGGTGGGGTATCTTC (SEQ ID NO:104). Thisfragment was cloned into the vector pCR2.1 TOPO (Invitrogen) accordingto the manufacturer's instructions. A clone containing the GAL4 gene(pCR2.1 TOPOGAL4) was digested with Hind III and the 2.7 kb GAL4fragment gel purified and ligated to the large fragment of pGADT7 thathad been digested with Hind III, treated with calf intestinalphosphotase and gel purified. Variants of the GAL4 gene were created byQuikchange reactions (Stratagene), carried out according to themanufacturers instructions, on pCR2.1 using primers listed in thesupplementary information. GAL4 mutants were cloned into pGADT7 in thesame manner as the wildtype GAL4 gene. All final constructs wereconfirmed by DNA sequencing.

Yeast Media and Manipulations

S. cerevisiae strain MaV203, (Invitrogen) is MATα; leu2-3,112; trp1109;his3 Δ200;ade2-101;cyh2^(R);cyh1^(R);GAL4 Δ; gal80 Δ; GAL1::lacZ;HIS3UASGAL1::HIS3@LYS2; SPAL10UASGAL1::URA3. Yeast media were purchasedfrom Clontech, 5-FOA and X-GAL were from Invitrogen and 3-AT was fromBIO 101. YPER (Yeast Protein Extraction Reagent) and ONPG were purchasedfrom Pierce Chemicals. Plasmid transformations were performed by thePEG/Lithium actetate method (see, e.g., D. Burke, et al., (2000) Methodsin Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) and transformants selected on the appropriate syntheticcomplete dropout media. To test the phenotypes conferred by variousplasmid combinations on MaV203 yeast colonies from synthetic completedropout plates of each transformation were resuspended in 15 μL ofsterile water and streaked on the selective media of interest. Eachphenotype was confirmed with at least five independent colonies. X-GALassays were performed by the agarose overlay method. See, I. G.Serebriiskii, & E. A. Golemis, (2000), Uses of lacZ to study genefunction: evaluation of beta-galactosidase assays employed in the yeasttwo-hybrid system, Analytical Biochemistry 285:1-15. Briefly, coloniesor cell patches were lysed on agar plates by several additions ofneatchlorofom. After chloroform evaporation 1% agarose containing 0.25g/L of XGAL and buffered with 0.1 M Na₂PO₄ was applied to the platesurface. Once the agarose was set, plates were incubated at 37° C. for12 h. ONPG assays were carried out by inoculation of 1 mL of SD-leu,-trp in a 96 well block with a single colony and incubated at 30° C.with shaking. The OD₆₆₀ of 100 μL of cells, and several dilutions ofcells were recorded in parallel in a 96 well microtiter plate. Cells(100 μL) were mixed with a 100 μL of YPER:ONPG (1×PBS, 50% v/v YPER, 20mM MgCl₂, 0.25% v/v β-mercaptoethanol, and 3 mM ONPG) and incubated withshaking at 37° C. Upon color development, cells were pelleted bycentrifugation, the supernatant transferred to a clean 96 wellmicrotiter plate (Nunclon, cat. #167008), and the A420 recorded. Alldata shown are the mean of trials from at least 4 independent clones andthe error bars shown represent the standard deviation. ONPG hydrolysiswas calculated using the equation: beta-galactosidase units=1000.A420/(V.t.OD₆₆₀, where V is the volume of cells in milliliters, t is thetime of incubation in minutes. See, e.g., I. G. Serebriiskii, & E. A.Golemis, (2000), Uses of lacZ to study gene function: evaluation ofbeta-galactosidase assays employed in the yeast two-hybrid system,Analytical Biochemistry 285:1-15. One beta-galactosidase unitcorresponds to the hydrolysis of 1 μmol of ONPG per minute per cell.See, Serebriiskii and Golemis, supra. Spectrophotometric readings wereperformed on a SPECTRAmax190 plate reader.

Model Selections

Positive selections: Two overnight cultures were grown in SD-Leu, -Trp.One contained MaV203 harboring pEcTyrRS-tRNA_(CUA)/pGADGAL4(T44,R110TAG) and the other pA5-tRNASU3/pGADGAL4(T44, R110TAG). These cellswere harvested by centrifugation and resuspended in 0.9% NaCl byvortexing. The two cell solutions were then diluted to identical OD₆₆₀s.MaV203 harboring pEcTyrRS-tRNA_(CUA)/pGADGAL4(T44,R110TAG) were seriallydiluted over 7 orders of magnitude and each dilution was then mixed 1:1vol:vol with undiluted MaV203 harboring pA5-tRNA_(CUA)/pGADGAL4(T44,R110TAG) to afford defined ratios of cells containing active andinactive tyrosyl-tRNA synthetase. For each ratio a second serialdilution was performed in which the number of cells was decreased butthe ratio of cells harboring pEcTyrRS-tRNA_(CUA)/pGADGAL4(T44,R110TAG)and pA5-tRNA_(CUA)/pGADGAL4(T44,R110TAG) was maintained. These dilutionswere plated on SD-Leu, -trp, SD-Leu, -Trp, -URA and SD-Leu, -Trp,-His+50 mM 3-AT. After 60 h the number of colonies on each plate wascounted, using an Eagle Eye CCD camera (Stratagene), and the phenotypeof survivors were confirmed with a X-GAL beta-galactosidase assay. Cellsfrom several individual blue or white colonies were isolated and grownto saturation in SD-leu, -trp and the plasmid DNA isolated by standardmethods. The identity of the EcTyrRS variant was confirmed by DNAsequencing.

Negative selection: The model negative selection was performed in ananalogous manner to the positive selection except that MaV203 harboringpA5-tRNA_(CUA)/pGADGAL4(T44,R110TAG) were serially diluted and mixedwith a fixed density of MaV203 harboringpEcTyrRS-tRNA_(CUA)/pGADGAL4(T44,R110TAG). Cells were plated on SD-leu,-trp+0.1% 5-FOA, the number of colonies counted after 48 hours and theplates processed as described above.

The following oligonucleotides (Table 3) were used in combination withtheir reverse complements to construct site-directed mutants byQuikchange mutagenesis. The position of the mutation is denoted by boldtext.

TABLE 3 OLIGONUCLEOTIDES USED TO CONSTRUCT SITE-DIRECTED MUTANTS. AmberMutants Oligo Sequence L3TAG 5′-ATGAAGTAGCTGTCTTCTATCGAACAAGCATGCG-3′(SEQ ID NO: 66) I13TAG 5′-CGAACAAGCATGCGATTAGTGCCGACTTAAAAAG-3′(SEQ ID NO: 67) T44TAG 5′-CGCTACTCTCCCAAATAGAAAAGGTCTCCGCTG-3′(SEQ ID NO: 68) F68TAG 5′-CTGGAACAGCTATAGCTACTGATTTTTCCTCG-3′(SEQ ID NO: 69) R110TAG 5′-GCCGTCACAGATTAGTTGGCTTCAGTGGAGACTG-3′(SEQ ID NO: 70) V114TAG 5′-GATTGGCTTCATAGGAGACTGATATGCTCTAAC-3′(SEQ ID NO: 71) T121TAG 5′-GCCTCTATAGTTGAGACAGCATAGAATAATGCG-3′(SEQ ID NO: 72) I127TAG 5′-GAGACAGCATAGATAGAGTGCGACATCATCATCGG-3′(SEQ ID NO: 73) S131TAG 5′-GAATAAGTGCGACATAGTCATCGGAAGAGAGTAGTAG-3′(SEQ ID NO: 74) T145TAG 5′-GGTCAAAGACAGTTGTAGGTATCGATTGACTCGGC-3′(SEQ ID NO: 75) Permissive Site Mutants Oligo Sequence T44F5′-CGCTACTCTCCCCAAATTTAAAAGGTCTCCGCTG-3′ (SEQ ID NO: 76) T44Y5′-CGCTACTCTCCCCAAATATAAAAGGTCTCCGCTG-3′ (SEQ ID NO: 77) T44W5′-CGCTACTCTCCCCAAATGGAAAAGGTCTCCGCTG-3′ (SEQ ID NO: 78) T44D5′-CGCTACTCTCCCCAAAGATAAAAGGTCTCCGCTG-3′ (SEQ ID NO: 79) T44K5′-CGCTACTCTCCCCAAAAAAAAAAGGTCTCCGCTG-3′ (SEQ ID NO: 80) R110F5′-GCCGTCACAGATTTTTTGGCTTCAGTGGAGACTG-3′ (SEQ ID NO: 81) R110Y5′-GCCGTCACAGATTATTTGGCTTCAGTGGAGACTG-3′ (SEQ ID NO: 82) R110W5′-GCCGTCACAGATTGGTTGGCTTCAGTGGAGACTG-3′ (SEQ ID NO: 83) R110D5′-GCCGTCACAGATGATTTGGCTTCAGTGGAGACTG-3′ (SEQ ID NO: 84) R110K5′-GCCGTCACAGATAAATTGGCTTCAGTGGAGACTG-3′ (SEQ ID NO: 85)

Example 2 An Expanded Eucaryotic Genetic Code

A general and rapid route for the addition of unnatural amino acids tothe genetic code of Saccharomyces cerevisiae is described. Five aminoacids have been incorporated into proteins efficiently, with highfidelity, in response to the nonsense codon TAG. The side chains ofthese amino acids contain a keto group, which can be uniquely modifiedin vitro and in vivo with a wide range of chemical probes and reagents;a heavy atom-containing amino acid for structural studies; andphotocrosslinkers for cellular studies of protein interactions. Thismethodology not only removes the constraints imposed by the genetic codeon our ability to manipulate protein structure and function in yeast, itprovides a gateway to the systematic expansion of the genetic codes ofmulticellular eukaryotes.

Although chemists have developed a powerful array of methods andstrategies to synthesize and manipulate the structures of smallmolecules (see, e.g., E. J. Corey, & X.-M. Cheng, The Logic of ChemicalSynthesis (Wiley-Interscience, New York, 1995)), the ability torationally control protein structure and function is still in itsinfancy. Mutagenesis methods are limited to the common 20 amino acidbuilding blocks, although in a number of cases it has been possible tocompetitively incorporate close structural analogues of common aminoacids throughout the proteome. See, e.g., K. Kirshenbaum, et al.,(2002), Chem Bio Chem 3:235-7; and, V. Doring et al., (2001), Science292:501-4. Total synthesis (see, e.g., B. Merrifield, (1986), Science232:341-7 (1986)), and semi-synthetic methodologies (see, e.g., D. Y.Jackson et al., (1994) Science 266:243-7; and, P. E. Dawson, & S. B.Kent, (2000), Annual Review of Biochemistry 69:923-60, have made itpossible to synthesize peptides and small proteins, but have morelimited utility with proteins over 10 kilodaltons (kDa). Biosyntheticmethods that involve chemically acylated orthogonal tRNAs (see, e.g., D.Mendel, et al., (1995), Annual Review of Biophysics and BiomolecularStructure 24:435-462; and, V. W. Cornish, et al. (Mar. 31, 1995),Angewandte Chemie-International Edition in English 34:621-633) haveallowed unnatural amino acids to be incorporated into larger proteins,both in vitro (see, e.g., J. A. Ellman, et al., (1992), Science255:197-200) and in microinjected cells (see, e.g., see, e.g., D. A.Dougherty, (2000), Current Opinion in Chemical Biology 4:645-52).However, the stoichiometric nature of chemical acylation severely limitsthe amount of protein that can be generated. Thus, despite considerableefforts the properties of proteins, and possibly entire organisms, havebeen limited throughout evolution by the twenty genetically encodedamino acids (with the rare exceptions of pyrrolysine and selenocysteine(see, e.g., A. Bock et al., (1991), Molecular Microbiology 5:515-20;and, G. Srinivasan, et al., (2002), Science 296:1459-62)).

To overcome this limitation, new components were added to the proteinbiosynthetic machinery of the prokaryote Escherichia coli (E. coli)(e.g., L. Wang, et al., (2001), Science 292:498-500), which make itpossible to genetically encode unnatural amino acids in vivo. A numberof new amino acids with novel chemical, physical or biologicalproperties have been incorporated efficiently and selectively intoproteins in response to the amber codon, TAG. See, e.g., J. W. Chin etal., (2002), Journal of the American Chemical Society 124:9026-9027; J.W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin,et al., (2002), PNAS United States of America 99:11020-11024: and, L.Wang, & P. G. Schultz, (2002), Chem. Comm., 1:1-10. However, because thetranslational machinery is not well conserved between prokaryotes andeukaryotes, components of the biosynthetic machinery added to E. colicannot generally be used to site-specifically incorporate unnaturalamino acids into proteins to study or manipulate cellular processes ineukaryotic cells.

Thus, translational components were created that would expand the numberof genetically encoded amino acids in eukaryotic cells. Saccharomycescerevisiae was chosen as the initial eukaryotic host organism, becauseit is a useful model eukaryote, genetic manipulations are facile (see,e.g., D. Burke, et al., (2000), Methods in Yeast Genetics (Cold SpringHarbor Laboratory Press, Cold Spring. Harbor, N.Y.), and itstranslational machinery is highly homologous to that of highereukaryotes (see, e.g., T. R. Hughes, (2002), Funct. Integr. Genomics2:199-211). The addition of new building blocks to the S. cerevisiaegenetic code requires a unique codon, tRNA, and aminoacyl-tRNAsynthetase (‘aaRS’) that do not cross-react with any components of theyeast translational machinery (see, e.g., Noren et al., (1989) Science244:182; Furter (1998) Protein Sci. 7:419; and, Liu et al., (1999) PNASUSA 96:4780). One candidate orthogonal pair is the amber suppressortyrosyl-tRNA synthetase-tRNA_(CUA) pair from E. coli (see, e.g., H. M.Goodman, et al., (1968), Nature 217:1019-24; and, D. G. Barker, et al.,(1982), FEBS Letters 150:419-23). E. coli tyrosyl-tRNA synthetase(TyrRS) efficiently aminoacylates E. coli tRNA_(CUA) when both aregenetically encoded in S. cerevisiae but does not aminoacylate S.cerevisiae cytoplasmic tRNAs. See, e.g., H. Edwards, & P. Schimmel,(1990), Molecular & Cellular Biology 10:1633-41; and, H. Edwards, etal., (1991), PNAS United States of America 88:1153-6. In addition, E.coli tyrosyl tRNA_(CUA) is a poor substrate for S. cerevisiaeaminoacyl-tRNA synthetases (see, e.g., V. Trezeguet, et al., (1991),Molecular & Cellular Biology 11:2744-51.) but is processed and exportedfrom the nucleus to the cytoplasm (see, e.g., S. L. Wolin, & A. G.Matera, (1999) Genes & Development 13:1-10) and functions efficiently inprotein translation in S. cerevisiae. See, e.g., H. Edwards, & P.Schimmel, (1990) Molecular & Cellular Biology 10:1633-41; H. Edwards, etal., (1991), PNAS United States of America 88:1153-6; and, V. Trezeguet,et al., (1991), Molecular & Cellular Biology 11:2744-51. Moreover, E.coli TyrRS does not have an editing mechanism and therefore should notproofread an unnatural amino acid ligated to the tRNA.

To alter the amino acid specificity of the orthogonal TyrRS so that itaminoacylates tRNA_(CUA) with a desired unnatural amino acid and none ofthe endogenous amino acids, a large library of TyrRS mutants wasgenerated and subject to a genetic selection. On the basis of thecrystal structure of the homologous TyrRS from Bacillusstearothermophilus (see, e.g., P. Brick, et al., (1989), Journal ofMolecular Biology 208:83) five residues ((B. stearothermophilus, FIG. 7,Panel A)) in the active site of E. coli. TyrRS that are within 6.5 Å ofthe para position of the aryl ring of bound tyrosine were mutated. Forexample, to create the EcTyrRS library of mutants the five positionstargeted for mutation were first converted to alanine codons to producethe A5RS gene. This was split between two plasmids at a unique Pst Isite in the gene. The library was created essentially as described bytechniques known in the art (see, e.g., Stemmer et al., (1993)Biotechniques 14:256-265). One plasmid contains the 5′ half of the A5RSgene, the other plasmid contains the 3′ half of the A5RS gene.Mutagenesis was performed on each fragment by PCR with oligonucleotideprimers for the amplification of the whole plasmid. The primers aredoped, containing NNK (N=A+G+T+C and K=G+T) and Bsa I restrictionendonuclease recognition sites. Digestion with Bsa I and ligationyielded two circular plasmids, each containing mutant copies of one halfof the EcTyrRS gene. The two plasmids were then digested with Pst I andassembled into a single plasmid by ligation, leading to assembly of thefull-length mutant genes. The mutant EcTyrRS genes were excised fromthis plasmid and ligated into pA5RS/tRNA_(CUA) between EcoR I and Not Isites. The library was transformed into S. cerevisiae Mav203: pGADGAL4(2TAG) using the PEG-lithium acetate method yielding ˜10⁸ independenttransformants.

A selection strain of S. cerevisiae [MaV203: pGADGAL4 (2 TAG) (see,e.g., M. Vidal, et al., (1996), PNAS United States of America93:10321-6; M. Vidal, et al., (1996), PNAS United States of America93:10315-201 and, Chin et al., (2003) Chem. Biol. 10:511)] wastransformed with the library to afford 10⁸ independent transformants andgrown in the presence of 1 mM unnatural amino acid (FIG. 8, Panel C).Suppression of two permissive amber codons in the transcriptionalactivator GAL4 leads to the production of full-length GAL4 and thetranscriptional activation of the GAL4-responsive HIS3, URA3, and lacZreporter genes (FIG. 8, Panel A). For example, the permissive cdons arefor T44 and R110 of Gal4. Expression of HIS3 and URA3 in media lackinguracil (-ura), or containing 20 mM 3-aminotriazole (see, e.g., G. M.Kishore, & D. M. Shah, (1988), Annual Review of Biochemistry 57, 627-63)(3-AT, a competitive inhibitor of the His3 protein) and lackinghistidine (-his), allows clones expressing active aaRS-tRNA_(CUA) pairsto be positively selected. If a mutant TyrRS charges the tRNA_(CUA) withan amino acid, then the cell biosynthesizes histidine and uracil andsurvives. Surviving cells were amplified in the absence of 3-AT andunnatural amino acid to remove full-length GAL4 from cells thatselectively incorporate the unnatural amino acid. To remove clones thatincorporate endogenous amino acids in response to the amber codon, cellswere grown on media containing 0.1% 5-fluorootic acid (5-FOA) butlacking the unnatural amino acid. Those cells expressing URA3, as aresult of suppression of the GALA amber mutations with natural aminoacids, convert 5-FOA to a toxic product, killing the cell. See, e.g., J.D. Boeke, et al., (1984), Molecular & General Genetics 197:345-6.Surviving clones were amplified in the presence of unnatural amino acidand reapplied to the positive selection. The lacZ reporter allows activeand inactive synthetase-tRNA pairs to be discriminated colorometrically(FIG. 8, Panel B).

With the use of this approach, five novel amino acids with distinctsteric and electronic properties (FIG. 7, Panel B) were independentlyadded to the genetic code of S. cerevisiae. These amino acids includep-acetyl-L-phenylalanine (1), p-benzoyl-L-phenylalanine (2),p-azido-L-phenylalanine (3), O-methyl-L-tyrosine (4), andp-iodo-L-phenylalanine (5) (indicated by the numbers in FIG. 7, PanelB). The unique reactivity of the keto functional group ofp-acetyl-L-phenylalanine allows selective modification of proteins withan array of hydrazine- or hydroxylamine-containing reagents in vitro andin vivo (See, e.g., V. W. Cornish, et al., (Aug. 28, 1996), Journal ofthe American Chemical Society 118:8150-8151; and, Zhang, Smith, Wang,Brock, Schultz, in preparation). The heavy atom ofp-iodo-L-phenylalanine can prove useful for phasing x-ray structure data(with the use of multiwavelength anomalous diffraction). Thebenzophenone and phenylazide side chains of p-benzoyl-L-phenylalanineand p-azido-L-phenylalanine allow efficient in vivo and in vitrophotocrosslinking of proteins (see e.g., Chin et al., (2002) J. Am.Chem. Soc., 124:9026; Chin and Schultz, (2002) Chem. Bio. Chem. 11:1135;and, Chin et al., (2002) PNAS, USA 99:11020). The methyl group ofO-methyl-L-tyrosine can be readily substituted with an isotopicallylabeled methyl group as a probe of local structure and dynamics with theuse of nuclear magnetic resonance and vibrational spectroscopy. Afterthree rounds of selection (positive-negative-positive), several colonieswere isolated whose survival on -ura or on 20 mM 3-AT-his media wasstrictly dependent on the addition of the selected unnatural amino acid.See, FIG. 8, Panel D. The same clones were blue on x-gal only in thepresence of 1 mM unnatural amino acid. These experiments demonstratethat the observed phenotypes result from the combination of the evolvedaminoacyl-tRNA synthetase-tRNA_(CUA) pairs and their cognate amino acids(see, Table 4).

For example, to select mutant synthetases, cells (˜10⁹) were grown for 4hours in liquid SD-leu, -trp+1 mM amino acid. Cells were then harvestedby centrifugation, resuspended in 0.9% NaCl, and plated on SD-leu, -trp,-his+20 mM 3-AT, +1 mM unnatural amino acid or SD-leu, -trp, -ura, +1 mMunnatural amino acid. After 48 to 60 hours at 30° C. the cells werescraped from the plates into liquid SD-leu, -trp and grown for 15 hoursat 30° C. Cells were harvested by centrifugation, resuspended in 0.9%NaCl and plated on SD-leu, -trp+0.1% 5-FOA. After 48 hours at 30° C.cells were scraped into liquid SD-leu, -trp+1 mM unnatural amino acidand grown for 15 hours. Cells were then harvested by centrifugation,resuspended in 0.9% NaCl, and plated on SD-leu, -trp, -his+20 mM 3-AT,+1 mM unnatural amino acid or SD-leu, -trp, -ura, +1 mM unnatural aminoacid. To screen phenotypes of selected cells, colonies (192) from eachselection were transferred to wells of 96 well blocks containing 0.5 mLof SD-leu, -trp and grown at 30° C. for 24 hours. Glycerol (50% v/v; 0.5mL) was added to each well, and the cells replica plated onto agar(SD-leu, -trp; SD-leu, -trp, -his, +20 mM 3-AT; SD-leu, -trp, -ura) inthe presence or absence of 1 mM unnatural amino acid. X-Gal assays wereperformed on SD-leu, -trp plates using the agarose overlay method.

To further demonstrate that the observed phenotypes are due tosite-specific incorporation of the unnatural amino acids by theorthogonal mutant TyrRS/tRNA pairs, mutants of human superoxidedismutase 1 (hSOD) (see, e.g., H. E. Parge, et al., (1992), PNAS UnitedStates of America 89:6109-13) containing each unnatural amino acid weregenerated and characterized.

For example, the addition of DNA encoding a C-terminal hexahistidinetag, and mutation of the codon for Trp 33 to an amber codon in the humansuperoxide dismutase gene was performed by overlap PCR using PS356(ATCC) as a template. hSOD (Trp 33 TAG) HIS was cloned between the GAL1promoter and CYC1 terminator from pYES2.1 (Invitrogen, Carlsbad, Calif.USA). Mutant synthetase and tRNA genes on pECTyrRS-tRNA_(CUA) derivedplasmids were co-transformed with pYES2.1 hSOD (Trp 33 TAG) HIS into thestrain InvSc (Invitrogen). For protein expression, cells were grown inSD-trp, -ura+ raffinose and expression induced at an OD₆₆₀ of 0.5 by theaddition of galactose. HSOD mutants were purified by Ni-NTAchromatography (Qiagen, Valencia, Calif., USA).

Production of hexa-histidine-tagged hSOD from a gene containing an ambercodon at position 33 was strictly dependent onp-acetylPheRS-1-tRNA_(CUA) and 1 mM p-acetyl-L-phenylalanine (<0.1% bydensitometry, in the absence of either component) (See FIG. 9).p-Acetyl-L-phenylalanine containing full-length hSOD was purified (e.g.,by Ni-NTA affinity chromatography) with a yield of 50 ng/mL, comparableto that purified from cells containing E. coli TyrRStRNA_(CUA). Forcomparison, wild type hSODHIS could be purified with a yield of 250ng/mL under identical conditions.

FIG. 9 illustrates protein expression of hSOD (33TAG)HIS in S.cerevisiae genetically encoding unnatural amino acids (as illustrated inFIG. 7, Panel B and indicated in FIG. 9 by their numbering in FIG. 7,Panel B). The top portion of FIG. 9 illustrates SDS-polyacrylamide gelelectrophoresis of hSOD purified from yeast in the presence (+) andabsence (−) of the unnatural amino acid indicated by the number, whichcorresponds to unnatural amino illustrated in FIG. 7, Panel B stain withCoomassie. Cells contain the mutant synthetase-tRNA pair selected forthe amino acid indicated. The center portion of FIG. 9 illustrates awestern blot probed with an antibody against hSOD. The bottom portion ofFIG. 9 illustrates a western blot probed with an antibody against theC-terminal His6 tag.

The identity of the amino acid incorporated was determined by subjectinga tryptic digest of the mutant protein to liquid chromatography andtandem mass spectrometry. For example, for mass spectrometry proteinbands were visualized by colloidal Coomassie stain. Gel bandscorresponding to wild-type and mutant SOD were excised frompolyacrylamide gels, sliced into 1.5-mm cubes, reduced and alkylated,then subjected to trypsin hydrolysis essentially as described. See,e.g., A. Shevchenko, et al., (1996), Analytical Chemistry 68, 850-858.Tryptic peptides containing the unnatural amino acid were analyzed bynanoflow reversed-phase HPLC/μESI/MS with an LCQ ion trap massspectrometer. Liquid chromatography tandem mass spectrometry (LC-MS/MS)analysis was performed on a Finnigan LCQ Deca ion trap mass spectrometer(Thermo Finnigan) fitted with a Nanospray HPLC (Agilent 1100 series).See, e.g., FIG. 10, Panels A-H.

The precursor ions corresponding to the singly and doubly charged ionsof the peptide Val-Y*-Gly-Ser-Ile-Lys (SEQ ID NO:87) containing theunnatural amino acids (denoted Y*) were separated and fragmented with anion trap mass spectrometer. The fragment ion masses could beunambiguously assigned, confirming the site-specific incorporation ofp-acetyl-L-phenylalanine (see, FIG. 10, Panel A). No indication oftyrosine or other amino acids in place of p-acetyl-L-phenylalanine wasobserved, and a minimum of 99.8% incorporation purity was obtained fromthe signal-to-noise ratio of the peptide spectra. Similar fidelity andefficiency in protein expression were observed when p-benzoylPheRS-1,p-azidoPheRS-1, O-meTyrRS-1, or p-iodoPheRS-1 was used to incorporatep-benzoyl-L-phenylalanine, p-azido-L-phenylalanine, O-methyl-L-tyrosine,or p-iodo-L-phenylalanine into hSOD (See, FIG. 9, and FIG. 10, PanelsA-H). In the experiments, p-Azido-L-phenylalanine is reduced top-amino-L-phenylalanine in sample preparation, and the latter isobserved in mass spectra. The reduction does not occur in vivo bychemical deriviatation of purified SOD containingp-azido-L-phenylalanine. In control experiments, hexa-histidine-taggedhSOD containing trypotophan, tyrosine, and leucine at position 33 wasprepared and subject to mass spectrometry (See, FIG. 10, Panels F, G andH). Ions containing amino acid 33 were clearly visible in the massspectra of these samples.

The independent addition of five unnatural amino acids to the geneticcode of S. cerevisiae demonstrates the generality of our method andsuggests that it can be applicable to other unnatural amino acidsincluding spin-labeled, metal-binding, or photoisomerizable amino acids.This methodology can allow the generation of proteins with new orenhanced properties as well as facilitate control of protein function inyeast. Moreover, in mammalian cells the E. coli tyrosyl-tRNA synthetaseforms an orthogonal pair with the B. stearothermophilus tRNA_(CUA). See,e.g., Sakamoto et al., (2002) Nucleic Acids Res. 30:4692. Therefore onecan use the aminoacyl-tRNA synthethases that have been evolved in yeastto add unnatural amino acids to the genetic codes of higher eukaryotes.

TABLE 4 SEQUENCES OF SELECTED AMINOACYL- TRNA SYNTHETASES. Residue # 37126 182 183 186 # clones Ec TyrRS Tyr Asn Asp Phe Leu p-IodoPheRS-1 ValAsn Ser Tyr Leu 1/8 p-IodoPheRS-2 Ile Asn Ser Met Leu 1/8 p-IodoPheRS-3Val Asn Ser Met Ala 6/8 p-OMePheRS-1 Val Asn Ser Met Leu  5/13p-OMePheRS-2 Thr Asn Thr Met Leu  1/13 p-OMePheRS-3 Thr Asn Thr Tyr Leu 1/13 p-OMePheRS-4 Leu Asn Ser Met Ser  1/13 p-OMePheRS-5 Leu Asn SerMet Ala  1/13 p-OMePheRS-6 Thr Asn Arg Met Leu  4/13 p-acetylPheRS-1^(a)Ile Asn Gly Met Ala 10/10 p-benzoylPheRS-1 Gly Asn Gly Phe Ala 1/2p-benzoylPheRS-2 Gly Asn Gly Tyr Met 1/2 p-azidoPheRS-1 Leu Asn Ser MetAla 1/6 p-azidoPheRS-2 Val Asn Ser Ala Ala 1/6 p-azidoPheRS-3 Leu AsnSer Ala Ala 1/6 p-azidoPheRS-4 Val Asn Ser Ala Val 1/6 p-azidoPheRS-5Ile Asp Asn Phe Val 1/6 p-azidoPheRS-6 Thr Asn Ser Ala Leu 1/6 ^(a)Theseclones also contain a Asp165Gly mutation

Example 3 Adding Amino Acid with Novel Reactivity to the Genetic Code ofEukaryotes

A site-specific, fast, reliable, and irreversible method ofbioconjugation to proteins based on a [3+2] cycloaddition isdemonstrated. There is a considerable need for chemical reactions thatmodify proteins under physiological conditions in a highly selectivefashion. See, e.g., Lemineux, & Bertozzi, (1996) TIBTECH, 16:506-513.Most reactions currently used for the selective modification of proteinsinvolve covalent bond formation between nucleophilic and electrophilicreaction partners, e.g. the reaction of α-haloketones with histidine orcysteine side chains. Selectivity in these cases is determined by thenumber and accessibility of the nucleophilic residues in the protein. Inthe case of synthetic or semisynthetic proteins, other more selectivereactions can be used such as the reaction of an unnatural keto-aminoacid with hydrazides or aminooxy compounds. See, e.g., Cornish, et al.,(1996) Am. Chem. Soc., 118:8150-8151; and, Mahal, et al., (1997)Science, 276:1125-1128. Recently, it has been possible to geneticallyencode unnatural amino acids (see, e.g., Wang, et al., (2001) Science292:498-500; Chin, et al., (2002) Am. Chem. Soc. 124:9026-9027; and,Chin, et al., (2002) Proc. Natl. Acad. Sci., 99:11020-11024), includingketone containing amino acids (see, e.g., Wang, et al., (2003) Proc.Natl. Acad. Sci., 100:56-61; Zhang, et al., (2003) Biochemistry,42:6735-6746; and, Chin, et al., (2003) Science, in press), in bacteriaand yeast using orthogonal tRNA-synthetase pairs with altered amino acidspecificities. This methodology has made possible the selective labelingof virtually any protein with a host of reagents including fluorophores,crosslinking agents and cytotoxic molecules.

A highly efficient method for the selective modification of proteins isdescribed, which involves the genetic incorporation of azide oracetylene containing unnatural amino acids into proteins in response to,e.g., the amber nonsense codon, TAG. These amino acid side chains canthen be modified by a Huisgen [3+2] cycloaddition reaction (see, e.g.,Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991) Ed. Trost,B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in 1,3-DipolarCycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, New York, p.1-176) with alkynyl (acetylene) or azide derivatives, respectively.Because this method involves a cycloaddition rather than a nucleophilicsubstitution, proteins can be modified with extremely high selectivity(another method that can be used is the ligand exchange on a bisarseniccompound with a tetracysteine motif, see, e.g., Griffin, et al., (1998)Science 281:269-272). This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Indeed, Finn and coworkers have shown that thisazide-alkyne [3+2] cycloaddition can be conducted on the surface of anintact cowpea mosaic virus. See, e.g., Wang, et al., (2003) J. Am. Chem.Soc., 125:3192-3193. For another recent example of the electrophilicintroduction of an azido group into a protein and a subsequent [3+2]cycloaddition, see, e.g., Speers, et al., (2003) J. Am. Chem. Soc.,125:4686-4687.

In order to selectively introduce either the alkynyl (acetylene) orazide functional group into eukaryotic proteins at unique sites, evolvedorthogonal TyrRS/tRNA_(CUA) pairs were generated in yeast thatgenetically encode the acetylene and azido amino acids, FIGS. 11, 1 and2, respectively. The resulting proteins can be efficiently andselectively labeled with fluorophores in a subsequent cycloadditionreaction under physiological conditions.

Previously, an E. coli tyrosyl tRNA-tRNA synthetase pair wasdemonstrated as being orthogonal in yeast, i.e., neither the tRNA northe synthetase cross react with the endogenous yeast tRNA orsynthetases. See, e.g., Chin, et al., (2003) Chem. Biol., 10:511-519.This orthogonal tRNA-synthetase pair has been used to selectively andefficiently incorporate a number of unnatural amino acids in yeast inresponse to the TAG codon (e.g., Chin, et al., (2003) Science, inpress). In order to alter the amino acid specificity of the E. colityrosyl-tRNA synthetase to accept amino acid 1 or 2 of FIG. 11, alibrary of ˜10⁷ mutants was generated by randomizing the codons forTyr³⁷, Asn¹²⁶, Asp¹⁸², Phe¹⁸³, and Leu¹⁸⁶. These five residues werechosen based on a crystal structure of the homologous synthetase from B.stearothermophilus. To obtain a synthetase for which the particularamino acid serves as a substrate, a selection scheme was used in whichthe codons for Thr⁴⁴ and Arg¹¹⁰ of the gene for the transcriptionalactivator GAL4 were converted to amber nonsense codons (TAG). See, e.g.,Chin, et al., (2003) Chem. Biol., 10:511-519. Suppression of these ambercodons in the MaV203:pGADGAL4(2TAG) yeast strain leads to production offull length GAL4 (see, e.g., Keegan, et al., (1986) Science,231:699-704; and, Ptashne, (1988) Nature, 335:683-689) which in turndrives expression of the HIS3 and URA3 reporter genes. The latter geneproducts complement histidine and uracil auxotrophy allowing clonesharboring active synthetase mutants to be selected in the presence of 1or 2 of FIG. 11. Synthetases that load endogenous amino acids areremoved by growth on medium lacking 1 or 2 of FIG. 11 but containing5-fluoroorotic acid, which is converted into a toxic product by URA3. Bypassing the library through three rounds of selection (positive,negative, positive), we identified synthetases selective for 1 of FIG.11 (pPR-EcRS1-5) and for 2 of FIG. 11 (pAZ-EcRS1-6) as shown in Table 8.

All synthetases show a strong sequence similarity, including a conservedAsn¹²⁶, suggesting an important functional role for this residue.Surprisingly, the synthetases pPR-EcRS-2 and pAZ-EcRS-6, evolved to bind1 and 2 of FIG. 11 respectively, converged to the same sequence(Tyr³⁷→Thr³⁷, Asn¹²⁶→Asn¹²⁶, Asn¹⁸²→Ser¹⁸², and Phe¹⁸³→Ala¹⁸³,Leu¹⁸⁶→Leu¹⁸⁶). Both hydrogen bonds between the phenolic hydroxy groupof bound tyrosine and Tyr³⁷ and Asp¹⁸² are disrupted by mutations to Thrand Ser, respectively. Phe¹⁸³ is converted to Ala, possibly providingmore space for the accommodation of the unnatural amino acid. To confirmthe ability of this synthetase (and the other synthetases) to accepteither amino acid as a substrate selection strains harboring thesynthetase plasmids were grown on media lacking uracil (the same resultswere obtained for media lacking histidine) but supplemented with either1 or 2 of FIG. 11. Growth results revealed that four of the five alkynesynthetases were able to load both unnatural amino acids onto its tRNA.The azido synthetases seem to be more selective, since only pAZ-EcRS-6(which is identical with pPR-EcRS-2) was able to amino acylate its tRNAwith both 1 and 2 of FIG. 11. The fact that no growth was detected inthe absence of 1 or 2 of FIG. 11 suggests that the synthetases do notaccept any of the 20 common amino acids as a substrate. See FIG. 14.

For all further experiments pPR-EcRS-2 (pAZ-EcRS-6) was used, allowingone to control which unnatural amino acid is incorporated simply byadding either 1 or 2 of FIG. 11 to media containing the expressionstrain. For protein production the codon for the permissive residueTrp³³ of human superoxide dismutase-1 (SOD) fused to a C-terminal 6× Histag was mutated to TAG. For example, human superoxide dismutase (Trp³³TAG) HIS was cloned between the GAL1 promoter and CYC1 terminator frompYES2.1 (Invitrogen, Carlsbad, Calif. USA). Mutant synthetase and tRNAgenes on pECTyrRS-tRNA_(CUA) derived plasmids were co-transformed withpYES2.1 SOD(Trp³³ TAG) HIS into the strain InvSc (Invitrogen). Forprotein expression, cells were grown in SD-trp, -ura+ raffinose andexpression was induced at an OD₆₆₀ of 0.5 by the addition of galactose.Protein was expressed in the presence or absence of 1 mM 1 or 2 of FIG.11 and purified by Ni-NTA chromatography (Qiagen, Valencia, Calif.,USA).

Analysis by SDS-PAGE and Western blot revealed unnatural amino aciddependent protein expression with a fidelity of >99% as judged bydensitometry comparisons to protein expression in absence of 1 or 2 ofFIG. 11. See FIG. 12. To further confirm the identity of the amino acidincorporated, a tryptic digest was subjected to liquid chromatographyand tandem mass spectrometry.

For example, the wild-type and mutant hSOD were purified using nickelaffinity column and protein bands were visualized by colloidal Coomassiestain. Gels bands corresponding to wild-type and mutant SOD were excisedfrom polyacrylamide gels, sliced into 1.5-mm cubes, reduced andalkylated, then subjected to trypsin hydrolysis essentially asdescribed. See, e.g., Shevchenko, A et al., (1996) Anal. Chem.68:850-858. Tryptic peptides containing the unnatural amino acid wereanalyzed by nanoflow reversed-phase HPLC/μESI/MS with an LCQ ion trapmass spectrometer. See, FIG. 15, Panel A and B. Liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis was performed on a FinniganLCQ Deca ion trap mass spectrometer (Thermo Finnigan) fitted with aNanospray HPLC (Agilent 1100 series).

The precursor ions corresponding to the singly and doubly chargedprecursor ions of the peptide VY*GSIK (SEQ ID NO:87) containing theunnatural amino acid (denoted Y*) were separated and fragmented with anion trap mass spectrometer. The fragment ion masses could beunambiguously assigned, confirming the site-specific incorporation ofeach unnatural amino acid. LC MS/MS did not indicate incorporation ofany natural amino acid at this position. The signal-to-noise of thepeptide for all mutants were >1000 suggesting fidelity of incorporationbetter than 99.8%. See, FIG. 15, Panel A and B.

To demonstrate that small organic molecules can be conjugated toproteins by an azide-alkyne [3+2] cycloaddition reaction, the dyes 3-6indicated in FIG. 13, Panel A, which contain either an acetylenic or anazido group and bear a dansyl or fluoresceine fluorophore, weresynthesized (see Example 5 herein). The cycloaddition itself was carriedout with 0.01 mM protein in phosphate buffer (PB), pH 8, in the presenceof 2 mM 3-6 indicated in FIG. 13, Panel A, 1 mM CuSO₄, and ˜1 mg Cu-wirefor 4 hours at 37° C. (see FIG. 13, Panel B).

For example, to 45 μL of protein in PB buffer (pH=8) was added 1 μL ofCuSO₄ (50 mM in H₂O), 2 μL of dye (50 mM in EtOH), 2 μL oftris(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)amine (50 mM in DMSO), and Cuwire. After 4 hours at room temperature or 37° C. or overnight at 4° C.,450 μL H₂O were added and the mixture was spun through a dialysismembrane (10 kDa cut off). After washing the supernatant with 2×500 μLby centrifugation, the solution was brought to a volume of 50 mL. Asample of 20 mL was analyzed by SDS-PAGE. Occasionally remaining dyecould be removed from the gel by soaking in H₂O/MeOH/AcOH (5:5:1)overnight. The use of tris(carboxyethyl)phosphine as the reducing agentgenerally led to less efficient labeling. In contrast to earlierobservations (e.g., Wang, Q. et al., (2003) J. Am. Chem. Soc.125:3192-3193), the presence or absence of the tris(triazolyl)amineligand did not have a substantial influence on the outcome of thereaction.

After dialysis the labeled proteins were then analyzed by SDS-PAGE andin-gel imaged using a densitometer in case of the dansyl dyes 3-4indicated in FIG. 13, Panel A (λ_(ex)=337 nm, λ_(em)=506 nm) or aphosphorimager in the case of the fluoresceine dyes 5-6 indicated inFIG. 13, Panel A (λ_(ex)=483 nm, λ_(em)=516 nm). See, e.g., Blake,(2001) Curr. Opin. Pharmacol., 1:533-539; Wouters, et al., (2001) Trendsin Cell Biology 11:203-211; and, Zacharias, et al., (2000) Curr. Opin.Neurobiol., 10:416-421. The labeled proteins were characterized by LCMS/MS analysis of tryptic digests showing site-specific attachment ofthe fluorophores and the conversion was 75% on average (e.g., asdetermined by comparison of A₂₈₀/A₄₉₅ values for SOD labeled with 5 or 6indicated in FIG. 13, Panel A). The selectivity of this bioconjugationis verified by the fact that there was no observable reaction between 3indicated in FIG. 13, Panel A and alkyne protein or 4 indicated in FIG.13, Panel A and azido protein.

TABLE 8 EVOLVED SYNTHETASES. pPR-EcRS selected for 1 and pAZ-EcRSselected for 2 (as indicated in FIG. 11) synthetase 37 126 182 183 186wild type Tyr Asn Asp Phe Leu pPR-EcRS-1 Gly Asn Ser Met Leu pPR-EcRS-2Thr Asn Ser Ala Leu pPR-EcRS-3 Ser Asn Thr Met Val pPR-EcRS-4 Ala AsnSer Tyr Leu pPR-EcRS-5 Ala Asn Thr Met Cys pAZ-EcRS-1 Leu Asn Ser MetAla pAZ-EcRS-2 Val Asn Ser Ala Ala pAZ-EcRS-3 Leu Asn Ser Ala AlapAZ-EcRS-4 Val Asn Ser Ala Val pAZ-EcRS-5 Ile Asp Asn Phe Val pAZ-EcRS-6Thr Asn Ser Ala Leu

Example 4 Synthesis of an Alkyne Amino Acid

In one aspect of the invention, the invention provides alkynyl aminoacids. An example of a structure of the alkynyl amino acid isillustrated by Formula IV:

An alkyne amino acid is typically any structure having Formula IV, whereR₁ is a substituent used in one of the twenty natural amino acids and R₂is an alkynyl substituent. For example, 1 in FIG. 11 illustrates thestructure of para-propargyloxyphenylalanine. p-Propargyloxyphenylalaninecan be synthesized, e.g., as outline below. In this embodiment, thesynthesis of p-propargyloxyphenylalanine can be completed in three stepsstarting from the commercially available N-Boc-tyrosine.

For example, N-tert-butoxycarbonyl-tyrosine (2 g, 7 mmol, 1 equiv.) andK₂CO₃ (3 g, 21 mmol, 3 equiv.) were suspended in anhydrous DMF (15 mL).Propargyl bromide (2.1 mL, 21 mmol, 3 equiv., 80% solution in toluene)was slowly added and the reaction mixture was stirred for 18 hours atroom temperature. Water (75 mL) and Et₂O (50 mL) were added, the layerswere separated and the aqueous phase was extracted with Et₂O (2×50 mL).The combined organic layers were dried (MgSO₄) and the solvent wasremoved under reduced pressure. The product was obtained as a yellow oil(2.3 g, 91%) and used in the next step without further purification. TheBoc-protected product is illustrated below as chemical structure 8:

2-tert-Butoxycarbonylamino-3-[4-(prop-2-ynyloxy)phenyl]-propionic acidpropargyl ester

Acetyl chloride (7 mL) was added carefully to methanol (60 mL) at 0° C.to give a 5 M solution of anhydrous HCl in MeOH. The product of theprevious step (2 g, 5.6 mmol) was added and the reaction was stirred for4 hours while it was allowed to warm to ambient temperature. Afterremoving the volatiles under reduced pressure, a yellowish solid (1.6 g,98%) (see chemical structure 9) was obtained which was directly used inthe next step.

2-Amino-3-[4-(prop-2-ynyloxy)phenyl]-propionic acid propargyl ester

The propargyl ester (1.6 g, 5.5 mmol) from the previous step wasdissolved in a mixture of aqueous 2 N NaOH (14 mL) and MeOH (10 mL).After stirring for 1.5 h at room temperature, the pH was adjusted to 7by adding conc. HCl. Water (20 mL) was added and the mixture was kept at4° C. overnight. The precipitate was filtered, washed with ice-cold H₂O,and dried under vacuum yielding, 1.23 g (90%) of 1 in FIG. 11(2-Amino-3-phenylpropionic acid (1) (also known asp-propargyloxyphenylalanine) as a white solid. ¹H NMR (400 MHz, D₂O) (asthe potassium salt in D₂O) δ 7.20 (d, J=8.8 Hz, 2H), 6.99 (d, J=8.8 Hz,2H), 4.75 (s, 2H), 3.50 (dd, J=5.6, 7.2 Hz, 1H), 2.95 (dd, J=5.6, 13.6Hz, 1H), 2.82 (dd, J=7.2, 13.6 Hz, 1H); ¹³C NMR (100 MHz, D₂O) δ 181.3,164.9, 155.6, 131.4, 130.7, 115.3, 57.3, 56.1, 39.3; HRMS (CI) m/z220.0969 [C₁₂H₁₃NO₃ (M+1) requires 220.0968].

Example 5 Addition of Molecules to Proteins with an Unnatural Amino AcidThrough a [3+2] Cycloaddition

In one aspect, the invention provides methods and related compositionsof proteins comprising unnatural amino acids coupled to additionalsubstituent molecules. For example, additional substituents can be addedto an unnatural amino acid through a [3+2] cycloaddition. See, e.g.,FIG. 16. For example, the [3+2] cycloaddition of a desired molecule(e.g., that include a second reactive group, such as an alkyne triplebond or azido group) to a protein with an unnatural amino acid (e.g.,having a first reactive group, such as azido group or triple bond) canbe done following published conditions for the [3+2] cycloadditionreaction. For example, a protein comprising the unnatural amino acid inPB buffer (pH=8) is added to CuSO₄, the desired molecule, and Cu wire.After the mixture is incubated (e.g., about 4 hours at room temperatureor 37° C., or overnight at 4° C.), H₂O is added and the mixture isfiltered through a dialysis membrane. The sample can be analyzed for theaddition, e.g., by gel analysis.

Examples of such molecules include, but are not limited to, e.g., amolecule having a triple bond or azido group, such as molecules have thestructure of Formula 3, 4, 5, and 6 of FIG. 13, Panel A and the like.Furthermore, triple bonds or azido groups can be incorporated into thestructures of other molecules of interest, such as polymers (e.g.,poly(ethylene glycol) and derivatives), crosslinking agents, additionaldyes, photocrosslinkers, cytotoxic compounds, affinity labels, biotin,saccharides, resins, beads, a second protein or polypeptide, metalchelators, cofactors, fatty acids, carbohydrates, polynucleotides (e.g.,DNA, RNA, etc.), and the like, which then can also be used in [3+2]cycloadditions.

In one aspect of the invention, molecules having the Formula 3, 4, 5, or6 of FIG. 13, Panel A can be synthesized as described below. Forexample, an alkyne dye as shown in 3 of FIG. 13, Panel A and in chemicalstructure 3 below was synthesized by adding propargylamine (250 μL, 3.71mmol, 3 equiv.) to a solution of dansyl chloride (500 mg, 1.85 mmol, 1equiv.) and triethylamine (258 μL, 1.85 mmol, 1 equiv.) in CH₂Cl₂ (10mL) at 0° C. After stirring for 1 hour, the reaction mixture was warmedto room temperature and stirred for an additional hour. The volatileswere removed in vacuo and the crude product was purified bychromatography on silica gel (Et₂O/hexanes=1:1) yielding 3 of FIG. 13,Panel A (418 mg, 78%) as a yellow solid. The analytical data areidentical with those reported in the literature. See, for example,Bolletta, F et al., (1996) Organometallics 15:2415-17. An example of astructure of an alkyne dye that can be used in the invention is shown inchemical structure 3:

An azido dye as shown as shown in 4 of FIG. 13, Panel A and in chemicalstructure 4 below was synthesized by adding 3-azidopropylamine (e.g., asdescribed by Carboni, B et al., (1993) J. Org. Chem. 58:3736-3741) (371mg, 3.71 mmol, 3 equiv.) to a solution of dansyl chloride (500 mg, 1.85mmol, 1 equiv.) and triethylamine (258 μL, 1.85 mmol, 1 equiv.) inCH₂Cl₂ (10 mL) at 0° C. After stirring for 1 hour, the reaction mixturewas warmed to room temperature and stirred for an additional hour. Thevolatiles were removed in vacuo and the crude product was purified bychromatography on silica gel (Et₂O/hexanes=1:1) yielding 4 of FIG. 13,Panel A (548 mg, 89%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.55(d, J=8.4 Hz, 1H), 8.29 (d, J=8.8 Hz, 1H), 8.23 (dd, J=1.2, 7.2 Hz, 1H),7.56-7.49 (comp, 2H), 7.18 (d, J=7.6 Hz, 1H), 5.24 (br s, 1H), 3.21 (t,J=6.4 Hz, 2H), 2.95 (dt, J=6.4 Hz, 2H), 2.89 (s, 6H), 1.62 (quin, J=6.4Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 134.3, 130.4, 129.7, 129.4, 128.4,123.3, 118.8, 115.3, 48.6, 45.4, 40.6, 28.7 (not all signals ofquaternary carbon atoms are visible in the ¹³C NMR spectrum);_([AD1])HRMS (CI) m/z 334.1336 [C₁₅H₂₀N₅O₂S (M+1) requires 334.1332]. Anexample of a structure of an azido dye is shown in chemical structure 4:

An alkyne dye as shown in 5 of FIG. 13, Panel A and in chemicalstructure 5 below was synthesized by adding EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (83 mg,0.43 mmol, 1 equiv.) to a solution of fluoresceinamine (150 mg, 0.43mmol, 1 equiv.) and 10-undecynoic acid (79 mg, 0.43, 1 equiv.) inpyridine (2 mL) at room temperature. The suspension was stirredovernight and the reaction mixture was poured into H₂O (15 mL). Thesolution was acidified (pH<2) by adding conc. HCl. After stirring for 1h, the precipitate was filtered off, washed with H₂O (5 mL) anddissolved in small amount of EtOAc. Addition of hexanes led to theprecipitation of 5 of FIG. 13, Panel A as orange crystals, which werecollected and dried under vacuum (138 mg, 63%). The analytical data areidentical with those reported in the literature. See, e.g., Crisp, G.T.; & Gore, J. (1997) Tetrahedron 53:1505-1522. An example of astructure of an alkyne dye is shown in chemical structure 5:

An azido dye as shown in 6 of FIG. 13, Panel A and in chemical structure6 below was synthesized by adding EDCI (83 mg, 0.43 mmol, 1 equiv.) to asolution of fluoresceinamine (150 mg, 0.43 mmol, 1 equiv.) and4-(3-azidopropylcarbamoyl)-butyric acid (e.g., synthesized by reacting3-azidopropylamine with glutaric acid anhydride) (92 mg, 0.43, 1 equiv.)in pyridine (2 mL) at room temperature. The suspension was stirred overnight and the reaction mixture was poured in H₂O (15 mL). The solutionwas acidified (pH<2) by adding conc. HCl. After stirring for 1 hour, theprecipitate was filtered off, washed with 1 N HCl (3×3 mL) and wasdissolved in a small amount of EtOAc. Addition of hexanes led to theprecipitation of 6 of FIG. 13, Panel A as orange crystals, which werecollected and dried under vacuum (200 mg, 86%). ¹H NMR (400 MHz, CD₃OD)δ 8.65 (s, 1H), 8.15 (d, J=8.4 Hz, 1H), 7.61-7.51 (comp, 2H), 7.40 (d,J=8.4 Hz, 1H), 7.35 (br s, 2H), 7.22-7.14 (comp, 2H), 6.85-6.56 (comp,3H), 3.40-3.24 (comp, 4H), 2.54 (t, J=7.2 Hz, 2H), 2.39-2.30 (comp, 2H),2.10-1.99 (comp, 2H), 1.82-1.72 (comp, 2H); ¹³C NMR (100 MHz, CD₃OD) δ175.7, 174.4, 172.4, 167.9, 160.8, 143.0, 134.3, 132.9, 131.8, 129.6,124.4, 123.3, 121.1, 118.5 103.5, 50.2, 38.0, 37.2, 36.2, 29.8, 22.9(not all signals of quaternary carbon atoms are visible in the ¹³C NMRspectrum); HRMS (CI) m/z 544.1835 [C₂₈H₂₅N₅O₇ (M+1) requires 544.1827].An example of a structure of an azido dye is shown in chemical structure6:

In one embodiment, a PEG molecule can also be added to a protein with anunnatural amino acid, e.g., an azido amino acid or a propargyl aminoacid. For example, a propargyl amide PEG (e.g., illustrated in FIG. 17,Panel A) can be added to a protein with an azido amino acid through a[3+2] cycloaddition. See e.g., FIG. 17, Panel A. FIG. 17, Panel Billustrates a gel analysis of a protein with an added PEG substituent.

In one aspect of the invention, a propargyl amide PEG (e.g., illustratedin FIG. 17, Panel A) can be synthesized as described below. For example,a solution of propargylamine (30 μL) in CH₂Cl₂ (1 mL) was added to the20 kDa PEG-hydroxysuccinimide ester (120 mg, purchased from Nektar). Thereaction was stirred for 4 hours at room temperature. Then Et₂O (10 mL)was added, the precipitate was filtered off, and was twicerecrystallized from MeOH (1 mL) by addition of Et₂O (10 mL). The productwas dried under vacuum furnishing a white solid (105 mg, 88% yield).See, e.g., FIG. 17, Panel C.

Example 6 Exemplary O-RSs and O-tRNAs

An exemplary O-tRNA comprises SEQ ID NO.:65 (See, Table 5). ExampleO-RSs include SEQ ID NOs.: 36-63, 86 (See, Table 5). Examples ofpolynucleotides that encode O-RSs or portions thereof (e.g., the activesite) include SEQ ID NOs.: 3-35. In addition, exemplary amino acidchanges of O-RSs are indicated in Table 6.

TABLE 6 Evolved EcTyrRS Variants Residue # 37 126 182 183 186Representation Ec TyrRS Tyr Asn Asp Phe Leu p-iodoPheRS-1 Val Asn SerTyr Leu 1/8  p-iodoPheRS-2 Ile Asn Ser Met Leu 1/8  p-iodoPheRS-3 ValAsn Ser Met Ala 6/8  OMeTyrRS-1 Val Asn Ser Met Leu 5/13 OMeTyrRS-2 ThrAsn Thr Met Leu 1/13 OMeTyrRS-3 Thr Asn Thr Tyr Leu 1/13 OMeTyrRS-4 LeuAsn Ser Met Ser 1/13 OMeTyrRS-5 Leu Asn Ser Met Ala 1/13 OMeTyrRS-6 ThrAsn Arg Met Leu 4/13 p-acetylPheRS-1 Ile Asn Gly Met Ala 4/4 p-acetylPheRS-1^(a) Ile Asn Gly Met Ala 10/10  p-benzoylPheRS-1 Gly AsnGly Phe Ala 1/2  p-benzoylPheRS-2 Gly Asn Gly Tyr Met 1/2 p-azidoPheRS-1 Leu Asn Ser Met Ala 1/6  p-azidoPheRS-2 Val Asn Ser AlaAla 1/6  p-azidoPheRS-3 Leu Asn Ser Ala Ala 1/6  p-azidoPheRS-4 Val AsnSer Ala Val 1/6  p-azidoPheRS-5 Ile Asp Asn Phe Val 1/6  p-azidoPheRS-6Thr Asn Ser Ala Leu 1/6  p-PR-EcRS-1 Gly Asn Ser Met Leu 1/10p-PR-EcRS-2 Thr Asn Ser Ala Leu 1/10 p-PR-EcRS-3 Ser Asn Thr Met Val1/10 p-PR-EcRS-4 Ala Asn Ser Tyr Leu 1/10 p-PR-EcRS-5 Ala Asn Thr MetCys 1/10 p-PR-EcRS-6 Thr Asn Thr Phe Met 1/10 p-PR-EcRS-7 Thr Asn SerVal Leu 1/10 p-PR-EcRS-8 Val Asn Ser Met Thr 1/10 p-PR-EcRS-9 Ser AsnSer Phe Leu 1/10 p-PR-EcRS-10 Thr Asn Thr Phe Thr 1/10 ^(a)These clonesalso contain a Asp165Gly mutation

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus describedherein can be used in various combinations. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication, patent, patentapplication, and/or other document were individually indicated to beincorporated by reference for all purposes.

TABLE 5 SEQ ID NO.: Label SEQUENCE SEQ ID E. coli wild-ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 1 type TyrRSGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGG (synthetase)CCCGATCGCGCTCTATTGCGGCTTCGATCCTACCGCTGACAGCTTGCAT polynucleotideTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCGAACAACTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGTTGCAGGGTTATGACTTCGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGT CTGATTTGCTGGAAATAASEQ ID E. coli wild- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALYCGFDPTADSLHLGHNO.: 2 type TyrRS LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV(synthetase) DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMAmino acid INKEAVKQRLNREDQGISFTEFSYNLLQGYDFACLNKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pOMe-1 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 3Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCpolynucleotide CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCcGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-2 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 4Synthetase gCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCpolynucleotide CCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-3 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 5Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCpolynucleotide CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-4 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAgCGGGGGCTGGTA NO.: 6Synthetase GCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCpolynucleotide CCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAgCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-5 ATGGCAAGCAGTAACTTGATTAAACAATTGCAAGAGCGGGGGCTGGTA NO.: 7Synthetase gCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCpolynucleotide CCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAGCCTGCTGCAGGGTTATACGATGGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTGTTTGGCCTGACCGTTCCGCTGATCACTAAAGCAGATGGCACCAAATTTGGTAAAACTGAAGGCGGCGCAGTCTGGTTGGATCCGAAGAAAACCAGCCCGTACAAATTCTACCAGTTCTGGATCAACACTGCGGATGCCGACGTTTACCGCTTCCTGAAGTTCTTCACCTTTATGAGCATTGAAGAGATCAACGCCCTGGAAGAAGAAGATAAAAACAGCGGTAAAGCACCGCGCGCCCAGTATGTACTGGCGGAGCAGGTGACTCGTCTGGTTCACGGTGAAGAAGGTTTACAGGCGGCAAAACGTATTACCGAATGCCTGTTCAGCGGTTCTTTGAGTGCGCTGAGTGAAGCGGACTTCGAACAGCTGGCGCAGGACGGCGTACCGATGGTTGAGATGGAAAAGGGCGCAGACCTGATGCAGGCACTGGTCGATTCTGAACTGCAACCTTCCCGTGGTCAGGCACGTAAAACTATCGCCTCCAATGCCATCACCATTAACGGTGAAAAACAGTCCGATCCTGAATACTTCTTTAAAGAAGAAGATCGTCTGTTTGGTCGTTTTACCTTACTGCGTCGCGGTAAAAAGAATTACTGTCTGATTTGC TGGAAATAA SEQ IDpOMe-6 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 8(active site) CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCpolynucleotide CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-7CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 9 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-8CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 10 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-9CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 11 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGTATGCCTGTGCGAACAAACAGTACG GTGTG SEQ ID pOMe-10CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 12 (active site)CTGGCGCAAGGCCCGATCGCACTCACTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCAGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTATGCCTGTCTGAACAAACAGTACG GTGTG SEQ ID pOMe-11CGGGGGCTGGTACCcCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 13 (active site)CTGGCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTATTGCCTGTTCGAACAAACAGTACG GTGTG SEQ ID pOMe-12CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 14 (active site)CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATTGCCTGTTTGAACAAACAGTACG GTGTG SEQ ID pOMe-13CGGGGGCTGGTACCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 15 (active site)CTGGCGCAAGGCCCGATCGCACTCGTGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTATTGCCTGTTTGAACAAACAGTACG GTGTG SEQ ID pOMe-14CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 16 (active site)CTGGCGCAAGGCCCGATCGCACTCTGGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATTGTTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATATGCGTGCCTGTGAGAACAAACAGTACG GTGTG SEQ IDp-acetylPhe-1 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 17(active site) CTGGCGCAAGGCCCGATCGCACTCATTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCpolynucleotide CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGGTCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTATGGCCTGTGCTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAATGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pBenzophenon-1CAGGTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCG NO.: 18 (active site)ATCGCACTCGGTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGG SynthetaseGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCA polynucleotideCAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTTTTGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pBenzophenone-2GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGGGTGTGGC NO.: 19 (activeTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTT site)ATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGT SynthetaseAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGA polynucleotideGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATGGTTATGCCTGTATGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAAT CAGGTG SEQ IDpAzidoPhe-1 GGGCTGGTAGCCCAGGTGACGGACGNAGAAGCGTTAGCAGAGCGACTG NO.: 20(active site) GCGCAAGGCCCGATCGCACTCCTTTGTGGCTTCGATCCTACCGCTGACASynthetase GCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGpolynucleotide CAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTATGGCCTGTGCGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCANAATCANGTG SEQ ID pAzidoPhe-2TTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCG NO.: 21 (active site)ATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGC SynthetaseCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGC polynucleotideGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTGCGGCCTGTGCGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCA GGTG SEQ IDpAzidoPhe-3 GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC NO.: 22(active site) CTGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTSynthetase TCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTpolynucleotide GCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAANAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGGCTGCCTGTGCGAACAAACAGTACGGNGNGGNGCTGCAAATTGGNGGTTCTGACCAGGGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTG CATCAAAATCAGGTG SEQ IDpAzidoPhe-4 GCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCT NO.: 23(active site) TCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTGSynthetase TGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTApolynucleotide GGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTGCGGCCTGTGTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATC ANGTG SEQ IDpAzidoPhe-5 GACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATCGCACTC NO.: 24(active site) ATTTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGCATCTTGTSynthetase TCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAAGCCGGTTpolynucleotide GCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATGATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAATTTTGCCTGTGTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGC ATCAGAATCAGGTG SEQ IDpAzidoPhe-6 CGACTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCG NO.: 25(active site) CTGACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCSynthetase TTCCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGpolynucleotide GGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-1CGGGGGCTGGTANCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 26 (propargyloxyCTGGCGCAAGGCCCGATCGCACTCGGGTGTGGCTTCGATCCTACCGCTG phenylalanineACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC synthetase)CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGT (active site)CTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACC SynthetaseGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCC polynucleotideCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTATGGCCTGTTTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGANCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-2CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 27 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGAACCTGANCCGTCGTCTGCATCAAAATCAAGTG SEQ ID pPR-EcRS-3CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAACGTTAGCAGAGCGA NO.: 28 (active site)CTGGCGCAAGGCCCGATCGCACTCTCTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCAGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGATGGCCTGTGTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-4CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 29 (active site)CTGGCGCAAGGCCCGATCGCACTCGCGTGCGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAGGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCTTATGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-5CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 30 (active site)CTGGCGCAAGGCCCGATCGCACTCGCGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGATGGCCTGTTGTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-6CGGGGGCTGGTACCCCAAGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 31 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCGCTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTTTGCCTGTATGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-7GTGACGGACGAGGAAGCGTTAGCAGAGCGACTGGCGCAAGGCCCGATC NO.: 32 (active site)GCACTCACGTGTGGCTTCGATCCTACCGCTGACAGCTTGCATTTGGGGC SynthetaseATCTTGTTCCATTGTTATGCCTGAAACGCTTCCAGCAGGCGGGCCACAA polynucleotideGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAATCTGCTGCAGGGTTATTCGGCTGCCTGTCTTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCG TCGTCTGCATCAGAATCAGGTGSEQ ID pPR-EcRS-8 CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGANO.: 33 (active site) CTGGCGCAAGGCCCGATCGCACTCGTTTGTGGCTTCGATCCTACCGCTGSynthetase ACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTCpolynucleotide CAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATTCGATGGCCTGTACGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-9CGGGGGCTGGTANCCCAAGTGACGGACGGGGAAGCGTTAGCAGAGCGA NO.: 34 (active site)CTGGCGCAAGGCCCGATCGCACTCAGTTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATCTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATAGTTTTGCCTGTCTGAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID pPR-EcRS-10CGGGGGCTGGTAGCCCAGGTGACGGACGAGGAAGCGTTAGCAGAGCGA NO.: 35 (active site)CTGGCGCAAGGCCCGATCGCACTCACGTGTGGCTTCGATCCTACCGCTG SynthetaseACAGCTTGCATTTGGGGCATCTTGTTCCATTGTTATGCCTGAAACGCTTC polynucleotideCAGCAGGCGGGCCACAAGCCGGTTGCGCTGGTAGGCGGCGCGACGGGTCTGATTGGCGACCCGAGCTTCAAAGCTGCCGAGCGTAAGCTGAACACCGAAGAAACTGTTCAGGAGTGGGTGGACAAAATCCGTAAGCAGGTTGCCCCGTTCCTCGATTTCGACTGTGGAGAAAACTCTGCTATCGCGGCCAATAATTATGACTGGTTCGGCAATATGAATGTGCTGACCTTCCTGCGCGATATTGGCAAACACTTCTCCGTTAACCAGATGATCAACAAAGAAGCGGTTAAGCAGCGTCTCAACCGTGAAGATCAGGGGATTTCGTTCACTGAGTTTTCCTACAACCTGCTGCAGGGTTATACGTTTGCCTGTACTAACAAACAGTACGGTGTGGTGCTGCAAATTGGTGGTTCTGACCAGTGGGGTAACATCACTTCTGGTATCGACCTGACCCGTCGTCTGCATCAGAATCAGGTG SEQ ID p-iodoPheRS-1MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH NO.: 36 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-iodoPheRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGHNO.: 37 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-iodoPheRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 38 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-1 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 39 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 40 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 41 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-4 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGHNO.: 42 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACSNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-5 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGHNO.: 43 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID OMeTyrRS-6 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 44 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYRMACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGH NO.: 45acetylPheRS-1 LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVSynthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREDQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH NO.: 46benzoylPheRS-1 LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVSynthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREDQGISFTEFSYNLLQGYGFACANKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p- MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGH NO.: 47benzoylPheRS-2 LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVSynthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREDQGISFTEFSYNLLQGYGYACMNKQYGVVLQIGGSDQ (aa)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-1MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH NO.: 48 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-2MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH NO.: 49 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-3MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALLCGFDPTADSLHLGH NO.: 50 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACANKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-4MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGH NO.: 51 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-5MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGH NO.: 52 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANDYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYNFACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID p-azidoPheRS-6MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGH NO.: 53 SynthetaseLVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWV Amino acidDKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-1 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALGCGFDPTADSLHLGHNO.: 54 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACLNKQYGVVLQIGGSDQ p-propargyloxy-WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTS phenylalaninePYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVL synthetaseAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-2 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 55 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSAACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-3 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHNO.: 56 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACVNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-4 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGHNO.: 57 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSYACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-5 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALACGFDPTADSLHLGHNO.: 58 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTMACCNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-6 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 59 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTFACMNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-7 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 60 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSVACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-8 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALVCGFDPTADSLHLGHNO.: 61 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSMACTNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-9 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALSCGFDPTADSLHLGHNO.: 62 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYSFACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID pPR-EcRS-10 MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALTCGFDPTADSLHLGHNO.: 63 Synthetase LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVAmino acid DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM (aa)INKEAVKQRLNREDQGISFTEFSYNLLQGYTFACTNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWKSEQ ID tRNA/Tyr AGCTTCCCGATAAGGGAGCAGGCCAGTAAAAAGCATTACCCCGTGGTG NO.: 64polynucleotide GGGTTCCCGAGCGGCCAAAGGGAGCAGACTCTAAATCTGCCGTCATCGACCTCGAAGGTTCGAATCCTTCCCCCACCACCA SEQ ID tRNA/TyrAGCUUCCCGAUAAGGGAGCAGGCCAGUAAAAAGCAUUACCCCGUGGU NO.: 65GGGGUUCCCGAGCGGCCAAAGGGAGCAGACUCUAAAUCUGCCGUCAUCGACCUCGAAGGUUCGAAUCCUUCCCCCACCACCA SEQ ID Amber5′-ATGAAGTAGCTGTCTTCTATCGAACAAGCATGCG-3′ NO.: 66 Mutants L3TAG SEQ IDAmber 5′-CGAACAAGCATGCGATTAGTGCCGACTTAAAAAG-3′ NO.: 67 Mutants I13TAGSEQ ID Amber 5′-CGCTACTCTCCCAAATAGAAAAGGTCTCCGCTG-3′ NO.: 68 MutantsT44TAG SEQ ID Amber 5′-CTGGAACAGCTATAGCTACTGATTTTTCCTCG-3′ NO.: 69Mutants F68TAG SEQ ID Amber 5′-GCCGTCACAGATTAGTTGGCTTCAGTGGAGACTG-3′NO.: 70 Mutants R110TAG SEQ ID Amber5′-GATTGGCTTCATAGGAGACTGATATGCTCTAAC-3′ NO.: 71 Mutants V114TAG SEQ IDAmber 5′-GCCTCTATAGTTGAGACAGCATAGAATAATGCG-3′ NO.: 72 Mutants T121TAGSEQ ID Amber 5′-GAGACAGCATAGATAGAGTGCGACATCATCATCGG-3′ NO.: 73 MutantsI127TAG SEQ ID Amber 5′-GAATAAGTGCGACATAGTCATCGGAAGAGAGTAGTAG-3′ NO.: 74Mutants S131TAG SEQ ID Amber 5′-GGTCAAAGACAGTTGTAGGTATCGATTGACTCGGC-3′NO.: 75 Mutants T145TAG SEQ ID Permissive5′-CGCTACTCTCCCCAAATTTAAAAGGTCTCCGCTG-3′ NO.: 76 Site Mutants T44FSEQ ID Permissive 5′-CGCTACTCTCCCCAAATATAAAAGGTCTCCGCTG-3′ NO.: 77Site Mutants T44Y SEQ ID Permissive5′-CGCTACTCTCCCCAAATGGAAAAGGTCTCCGCTG-3′ NO.: 78 Site Mutants T44WSEQ ID Permissive 5′-CGCTACTCTCCCCAAAGATAAAAGGTCTCCGCTG-3′ NO.: 79Site Mutants T44D SEQ ID Permissive5′-CGCTACTCTCCCCAAAAAAAAAAGGTCTCCGCTG-3′ NO.: 80 Site Mutants T44KSEQ ID Permissive 5′-GCCGTCACAGATTTTTTGGCTTCAGTGGAGACTG-3′ NO.: 81Site Mutants R110F SEQ ID Permissive5′-GCCGTCACAGATTATTTGGCTTCAGTGGAGACTG-3′ NO.: 82 Site Mutants R110YSEQ ID Permissive 5′-GCCGTCACAGATTGGTTGGCTTCAGTGGAGACTG-3′ NO.: 83Site Mutants R110W SEQ ID Permissive5′-GCCGTCACAGATGATTTGGCTTCAGTGGAGACTG-3′ NO.: 84 Site Mutants R110DSEQ ID Permissive 5′-GCCGTCACAGATAAATTGGCTTCAGTGGAGACTG-3′ NO.: 85Site Mutants R110K SEQ ID p-MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALICGFDPTADSLHLGH NO.: 86acetylPheRS-1 LVPLLCLKRFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVSynthetase DKIRKQVAPFLDFDCGENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQM Amino acidINKEAVKQRLNREGQGISFTEFSYNLLQGYGMACANKQYGVVLQIGGSDQ (aa)^(a)WGNITSGIDLTRRLHQNQVFGLTVPLITKADGTKFGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSGKAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVEMEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLF GRFTLLRRGKKNYCLICWK^(a)These clones also contain a Asp165G1y mutation

1. A method of producing in a eukaryotic cell at least one fluorescentprotein comprising at least one unnatural amino acid, the methodcomprising: growing, in an appropriate medium, a eukaryotic cell thatcomprises a nucleic acid that comprises at least one selector codon andencodes the fluorescent protein; wherein the eukaryotic cell comprises:an orthogonal tRNA (O-tRNA) that functions in the cell and recognizesthe selector codon; and an orthogonal aminoacyl tRNA synthetase (O-RS)as set forth in any one of SEQ ID NO: 36-47 or 86, wherein the O-RSpreferentially aminoacylates the O-tRNA with the unnatural amino acid,wherein the unnatural amino acid is selected from ap-acetyl-L-phenylalanine, a p-iodo-L-phenylalanine, anO-methyl-L-tyrosine, or a p-benzoyl-L-phenylalanine, and wherein saidfluorescent protein is produced when said unnatural amino acid is addedto said medium.
 2. The method of claim 1, wherein the O-tRNA comprises,is cellularly processed from, or is encoded by SEQ ID NO.: 64 or 65, ora complementary polynucleotide sequence thereof.
 3. The method of claim1, wherein the protein comprises a therapeutic protein, a diagnosticprotein, an industrial enzyme, or portion thereof.
 4. The method ofclaim 1, wherein the protein comprises a green fluorescent protein or aportion thereof.